Name: intraportal transplantation of pancreatic islets in mouse model
Uploaded: 2018-05-05T15:00:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

我们想感谢 Gundula Hertl 的支持。

本议定书的所有程序均经德国动物福利法和准则批准。考虑使用 10-到12周的 NMRI 裸鼠和 C57Bl/6 小鼠 (捐赠者: 老女性; 收件人: 男性) 贯穿整个实验。使用 NMRI 裸鼠为胰岛隔离。根据当地动物设施规定, 将小鼠置于规定的条件下。
1. 链脲佐菌素诱导糖尿病
<ol><li>4天, 用称重机测量10至12周的 NMRI 裸鼠和 C57Bl/6 小鼠的体重。</li>
	<li>根据测量的体重, 准备180毫克/千克剂量的链脲佐菌素在0.1 毫升生理盐水每只老鼠。 注: 链脲佐菌素应在使用和覆盖铝箔之前进行新的准备, 因为它是光敏的。</li>
	<li>注射自体和同种异体小鼠每一个剂量的0.1 毫升链脲佐菌素 (180 毫克/千克) 腹腔使用26克针。</li>
	<li>日-3,-2 和-1 收集2-4 µL 的血液通过穿刺的尾部静脉的非禁食小鼠。</li>
	<li>用每条试纸的2-4 µL 测定 NMRI 裸鼠和 C57Bl/6 小鼠的血糖 注意: 在这一点上, 80% 的老鼠应该成为糖尿病 (20 毫摩尔/升)。<ol>
<li>在胰岛移植时使用小鼠血糖水平达到20毫摩尔/升。</li>
	</ol>
</li>
</ol>2. 小鼠胰岛的分离
<ol><li>麻醉小鼠 (100 毫克/千克体重) 和甲苯噻嗪 (20 毫克/千克体重) 腹腔注射与26克针。 注: 氯胺酮和甲苯噻嗪的体积是根据动物的体重 (0.10 mL/10 g) 确定的。</li>
	<li>保持小鼠仰卧位, 用聚维酮碘溶液进行消毒。通过检查踏板反射来确认正确的安乐死。 注: 万一 C57Bl/6 剃了腹部区域。</li>
	<li>用细钳握住皮肤, 用剪刀做中线切口, 露出腹腔。将肠道放置在不育纱布压缩下的腹腔左侧和外腔。</li>
	<li>调整胃在另一个方向, 以找到胰腺和其他相关组织。</li>
	<li>到达主动脉, 做一个小切口排泄血液。使用无菌纱布进行血液吸收。</li>
	<li>小心地取出肠道。将胰腺与胃和脾脏分开。</li>
	<li>把胰脏放在新鲜的培养皿里。用镊子取出所有不需要的材料, 如脂肪。</li>
	<li>用4毫升的胶原酶填充5毫升的注射器, 用26克针 (股票溶液: 在汉克溶液的12毫升中用30毫克胶原酶) 注入胰腺。</li>
	<li>用剪刀将胰腺剁碎, 增加表面积, 改善消化, 转移到12毫升红帽管。 注: 汉克的平衡盐溶液: HBSS 10x (100 毫升), 青霉素-链霉素 (10 毫升), 庆大霉素 (1 毫升), 环丙沙星 (10 毫升), HEPES (35 毫升), 蒸馏 H2O (900 毫升)</li>
	<li>在颤抖的水浴中进行10分钟的消化, 37 摄氏度。在十五年代的固定时间间隔内, 对样品进行三次涡旋。</li>
	<li>用手大力混合消化的胰腺3分钟, 并将其放在冰上十年代。</li>
	<li>添加6毫升的冷汉克溶液, 以停止消化反应和离心机在 560 x g 在室温下3分钟。</li>
	<li>在室温下取出上清液, 在10毫升的帕克/胎小牛血清 (P/FCS) 培养基中溶解颗粒。 注: P/FCS 介质: TCM-199 10x (100 毫升), FCS (50 毫升), 青霉素-链霉素 (10 毫升), HEPES (20 毫升), 蒸馏 H2O (900 毫升)</li>
	<li>根据光镜下的形态学 (球状, 金黄色-褐色) 识别胰岛。避免带深色中心的小岛。在光显微镜下进行双硫腙染色, 检查纯度和活性。 注: 双硫腙与锌离子结合, 为胰岛提供红色染色。</li>
	<li>用1毫升的吸管在光显微镜下计数和精选胰岛, 将它们转移到含有4毫升 P/FCS 培养基的新鲜培养皿 (60 毫米 x 15 毫米)。 注: 培养皿中的P/FCS 介质的体积不应超过4毫升, 以确保氧气的可用性。</li>
	<li>将孤立的胰岛在4毫升的 P/FCS 培养基中过夜, 在37摄氏度的孵化器。</li>
</ol>3. 胰岛移植动物的制备
<ol><li>从37摄氏度孵化器中取出孤立的小岛, 并将它们放在流动工作台下。中心的胰岛与一个圆形摆动运动的培养皿。</li>
	<li>用0.1 毫升的 P/FCS 培养基填充1毫升注射器 (蓝色23克针)。然后, 在0.3 毫升的汉克溶液中添加350个胰岛。将注射器保持在垂直位置, 使小岛能够安定下来。</li>
	<li>将体积减小到0.05 毫升, 用褐色的26克针取代蓝色的23克针。对同一注射器, 增加0.15 毫升的 Ficoll (密度梯度培养基) 到顶端, 以达到最终体积0.2 毫升的注射器。避免气泡。</li>
	<li>将注射器保持在垂直位置, 稍微旋转, 以避免胰岛结块。胰岛将在2-3 分钟内在注射器的锥形处沉淀。</li>
	<li>测量受体小鼠的体重和血糖水平;血糖水平应约为20毫摩尔/升。</li>
	<li>麻醉小鼠 (100 毫克/千克体重) 和甲苯噻嗪 (20 毫克/千克体重) 腹腔注射26克针。</li>
	<li>将小鼠置于仰卧位, 放在暖热板上 (37 摄氏度)。通过捏脚趾来检查麻醉深度。</li>
	<li>用聚维酮碘溶液 (皮肤消毒剂) 清洁腹部区域。为防止眼部干燥, 请用眼膏保湿。 注: 万一 C57Bl/6 剃了腹部区域。</li>
</ol>4. 胰岛移植通过门静脉路线
<ol><li>0天, 从剖腹手术开始, 用钳夹住皮肤, 用剪刀做2-3 厘米长的中线切口。将无菌湿纱布放在伤口边缘周围, 保持湿润。</li>
	<li>将肠道置于左侧和腹腔外的无菌纱布压缩。 注意: 在整个过程中保持肠道湿润, 预热前0.9% 氯化钠溶液或不育纱布浸泡在生理盐水中。</li>
	<li>移动十二指肠并定位胰腺。通过将胰腺向上推, 找到位于腹侧部位的门静脉。使用手指和拇指暴露的门静脉, 一旦暴露, 刺穿它附近的肝脏。</li>
	<li>在1分钟内, 在放大镜的检查下缓慢和稳步地将胰岛 (0.2 毫升从步骤 3.4) 注入门静脉, 以避免胰岛的任何渗漏。 注: 注射后, 应始终检查注射器中的其余胰岛。在注射器中服用1毫升的 P/FCS 介质, 冲洗成新鲜的培养皿, 并在显微镜下计数。</li>
	<li>在穿刺部位放置一个不育的量规, 用食指 (穿透深度: 0.5-1 厘米) 施加压力, 以6分钟止血。 注: 注射量不应超过0.2 毫升。由于剪切应力, 体积和压力过大会破坏小岛。</li>
	<li>小心地将肠道、腹膜、腹部肌肉层和筋膜放回原位。</li>
	<li>使用镊子, 抬起皮肤, 用可吸收的丝缝线闭合肌肉切口, 上部皮肤层有2-3 个夹子。</li>
	<li>用皮肤消毒剂清洁表面, 应用伤口治疗, 并将老鼠放在单独的笼子里。伤口治疗提供了耐水性丙烯酸酯层, 防止浸渍和继发感染。 注意: 观察老鼠, 直到他们恢复足够的意识, 提供曲马多 (每只老鼠0.2 毫克) 连同液体。在本协议中, 小鼠快速从麻醉中恢复, 曲马多管理减轻了疼痛。7天后伤口就会愈合</li>
	<li>用普通的食物和水喂老鼠。食品和水的消耗和尿液排泄将增加后, 佐菌素注射液。</li>
	<li>每天在自体小鼠的第一周和每周一次测量血糖水平。在同种异体小鼠中, 每周三次测量血糖水平长达15天。</li>
</ol>

<ol>
	<li>McCall, M., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), a007823 (2012).</li><li>Shapiro, A. 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所有作者都宣称他们没有利益冲突。

本文对胰岛移植的门静脉路线进行了探讨。该协议在缓解糖尿病应激方面具有高效性, 为探索胰岛移植研究提供了深入的机会。该协议证实, 大约350只小岛能够逆转高血糖状态。此外, 所有的老鼠在手术过程中都没有死亡, 所有接受者的血糖控制都达到了。定期记录体重和血糖水平, 以反映移植胰岛逆转高血糖的能力、标准和功能。在 syngeneically 移植的小鼠中, 血糖水平每天在第一周和一周内测量一?...

胰岛移植是治疗1型糖尿病的一种很有前景的方法1。第一次尝试转移绵羊胰腺碎片到糖尿病患者是由沃森威廉斯在1893年进行。然而, 通过埃德蒙顿协议实现了临床胰岛移植的一个重大突破, 此后又制定了一系列国家计划2。过去, 在临床前模型中探讨了骨髓、网膜囊、肌内区、胃粘膜表面、脾、肾胶囊等几个部位, 但门静脉系统被认为是适宜有效的部位之一。对于临床程序3,4,5,6。
胰岛素的外源性管理可以用胰岛输液代替门静脉。这被认为是一个首选的网站, 因为氧气供应是可比的, 在当地的胰腺由于在下游的位置的汇合的门动脉和静脉。此外, 还有一个很大的表面积, 这样可以保留胰岛的三维结构, 并且可以方便地进行血管化5。在接受糖尿病的老鼠中, 2000 人或猪胰岛当量或350小鼠胰岛足以逆转高血糖状态7,8。钳水平被报告了15天在小鼠接受者模型的异种胰岛和在同种异体鼠-鼠模型, 并在120天的自体鼠对鼠移植。
与通过门静脉胰岛移植的疗效相关的因素是适当的麻醉、穿刺方法和止血9。麻醉可以诱导吸入 (5% 异氟烷) 或注射 (氯胺酮, 甲苯噻嗪, 或戊巴比妥), 这些物质往往结合。为了控制麻醉的深度和时间, 应注意鼠标的状态, 如粘膜的颜色、眼盖、角膜反射、呼吸速率和体温。重要的是要确保动物不挣扎, 它生存的行动。温度可由不同的加热装置维护, 如加热垫、红光灯泡、等.热热板已用于保持25-30 摄氏度之间的温度, 在鼠标和操作表之间, 防止发生低温。麻醉药的剂量是重要的, 因为所有这些药物都是由肝脏代谢的, 肝功能可能短暂紊乱的胰岛悬浮液注入.门静脉的理想穿刺点是第一和第二支流静脉的位置。
胰岛的数量和预期的注射量也会影响移植的结果, 因为过量的体积可能会增加剪切应力。0.2 毫升体积被认为适合胰岛移植入门静脉。穿刺伤口 (26 口径针) 诱导门静脉出血, 需要及时有效地停止。无菌纱布或手指可在穿刺部位施加最小压力, 以止血6分钟左右。结合, 门胰岛注射液是有效的, 并提供调节血糖水平的化学诱导糖尿病小鼠模型。

<table><tbody><tr><td>C57Bl/6 mice&amp;nbsp;</td><td>Charles River (Sulzfeld, Germany)</td><td></td><td>(10-12 weeks)</td></tr><tr><td>BALB/c mice</td><td>Charles River (Sulzfeld, Germany)</td><td></td><td>(10-12 weeks)</td></tr><tr><td>NMRI nude mice</td><td>Charles River (Sulzfeld, Germany)</td><td></td><td>(10-12 weeks)</td></tr><tr><td>Ketamine</td><td>Bela-pharm</td><td></td><td></td></tr><tr><td>Xylazine</td><td>CEVA TIERGESUNDHEIT GmbH</td><td></td><td></td></tr><tr><td>Syringe, 23G and 26G needle</td><td>BD</td><td></td><td></td></tr><tr><td>Petri Dish</td><td>Falcon</td><td>303800</td><td></td></tr><tr><td>NaCl</td><td>Carl Roth</td><td>351007</td><td></td></tr><tr><td>Sterile Gauze</td><td>Fuhrmann</td><td>7647-14-5</td><td></td></tr><tr><td>Shaking Water bath</td><td>Kotterman</td><td>1501</td><td></td></tr><tr><td>Scissors</td><td>Braun</td><td>1501</td><td></td></tr><tr><td>Glucose Meter&amp;nbsp;</td><td>One Touch</td><td></td><td></td></tr><tr><td>Warm Thermal Plate</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Braunol (povidone-iodide solution)</td><td>B.Braun Melsungen AG</td><td>3864154</td><td></td></tr><tr><td>Liposic Edo - Sprinkled Ointment (eye ointment)</td><td>Dr. G Mann Chem.-Pharm.&amp;nbsp; GmbH</td><td></td><td></td></tr><tr><td>Incubator</td><td>Fa. Roth</td><td></td><td></td></tr><tr><td>Flow Bench</td><td>Herdus</td><td></td><td></td></tr><tr><td>Ficoll</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>5-0 Silk Suture Vicryl</td><td>Braun</td><td></td><td>(7.5 *1.75mm)</td></tr><tr><td>Michel Clamps (clips)</td><td>Aesculap&amp;nbsp;</td><td>BN507R</td><td></td></tr><tr><td>P/FCS Solution</td><td>Gibco</td><td>21180-021</td><td></td></tr><tr><td>TCM-199 (10x)</td><td>Gibco</td><td>21180-021</td><td></td></tr><tr><td>FCS</td><td>Biowest</td><td>S1810-500</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Biochrom</td><td>L 1613</td><td></td></tr><tr><td>Gentamycin</td><td>Ratiopharm</td><td></td><td></td></tr><tr><td>Ciprofloxacin</td><td>Fresenius Kabi</td><td></td><td></td></tr><tr><td>Hank&#039;s Solution</td><td>Gibco</td><td>14060-040</td><td></td></tr><tr><td>Collagenase</td><td>Roche</td><td>11213865001</td><td></td></tr><tr><td>Streptozotocin</td><td>Calbiochem</td><td>572204</td><td></td></tr><tr><td>OPSITE-spray (wound healer)</td><td>Smith and nephew</td><td>66004978</td><td></td></tr><tr><td>Reugular food</td><td>Altromin</td><td></td><td></td></tr><tr><td>Head light LED 16042 (magnifying glass)</td><td>Eschenbach</td><td>16042</td><td></td></tr><tr><td>Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody</td><td>Millipore, Chemicon</td><td>CBL1337</td><td>Dilution (1:100)</td></tr><tr><td>Donkey anti-rat secondary antibody</td><td>Dianova</td><td>712095153</td><td>Dilution (1:400)</td></tr><tr><td>Polyclonal guinea pig anti-insulin primay antibody</td><td>Dako</td><td>A0564</td><td>Dilution (1:500)</td></tr><tr><td>Donkey anti-guinea pig secondary antibody</td><td>Dianova</td><td>706-259-148</td><td>Dilution (1:400)</td></tr></tbody></table>

intraportal transplantation of pancreatic islets in mouse model

胰岛移植降低高血糖是非常成功的啮齿动物与化学性糖尿病。实验性胰岛移植最常见的移植部位为肾囊。然而, 由于对胰岛与血液成分的相互作用知之较少, 因此在实验性胰岛移植中利用门静脉途径也有意义。
该协议表明了门静脉胰岛移植技术在 NMRI 裸鼠。腹腔注射佐菌素 (180 毫克/千克) 诱导受体小鼠高血糖。他们被认为是糖尿病在非空腹血糖水平大于20毫摩尔/升。在移植前的某一天, 用胶原酶消化法从供体胰腺中分离出小鼠胰岛;每位糖尿病患者至少使用350个胰岛。根据胰岛的分离率, 每个受体使用两个或更多的捐献小鼠。在隔夜文化在37°c 以后, 胰岛通过门静脉被管理入接受者肝脏。手术后, 小鼠被保护在红色 Makrolon 的房子和观察, 直到是清醒的。该协议维持血糖控制120天的自体小鼠和15天的同种异体小鼠。

研究了化学诱导的糖尿病小鼠移植胰岛的能力。胰岛移植通过门静脉系统的糖尿病小鼠模型。捐助者接受率为2:1。在自体小鼠模型中, 利用两只捐献小鼠获得350个胰岛。然后将胰岛移植到糖尿病小鼠体内。移植后1天观察到血糖水平的降低, 提示移植胰岛在降低血糖水平中的作用。链脲佐菌素增加了血糖水平高达约22毫摩尔/升。胰岛移植后, 血糖水平下降到4.6 毫摩尔/升 (1 天...

Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

Intraportal Transplantation of Pancreatic Islets in Mouse Model

胰岛移植是实现1型糖尿病血糖浓度的一种途径。本文主要研究了通过门静脉途径维持糖尿病小鼠正常血糖水平的移植技术。

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 ...

intraportal-transplantation-of-pancreatic-islets-in-mouse-model

Research

JoVE Journal

Medicine

9.4K 次观看.  Clinical Research Lab. 胰岛移植是实现1型糖尿病血糖浓度的一种途径。本文主要研究了通过门静脉途径维持糖尿病小鼠正常血糖水平的移植技术。

视频: Intraportal Transplantation of Pancreatic Islets in Mouse Model

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

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

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

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 ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

毛里岛皮下白脂肪组织（ISWAT）移植模型

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

尽量减少小鼠肝内胰岛移植期间输注后门静脉出血

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

小鼠骨髓胰岛移植的一种方法

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging

Source: Nilsson, J.et. al.&#160;<a href="https://www.jove.com/v/61234/" target="_blank">A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber</a>. J. Vis. Exp. (2020).
This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse&#8217;s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

移植胰岛:一种将胰岛植入小鼠眼前房进行体内成像的方法

Source: Nilsson, J.et. al.&#160;A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the ...

胰岛门静脉移植小鼠模型的研究

摘要

探索更多视频

此视频中的章节

小鼠胰岛的分离和包膜下肾移植的方法

补肾胶囊对糖尿病小鼠的胰岛移植

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

应用改良袖套技术的小鼠胰腺移植模型

毛里岛皮下白脂肪组织（ISWAT）移植模型

尽量减少小鼠肝内胰岛移植期间输注后门静脉出血

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

胰腺胰岛与小鼠分离的简单高效协议

小鼠骨髓胰岛移植的一种方法

移植胰岛:一种将胰岛植入小鼠眼前房进行体内成像的方法

胰岛门静脉移植小鼠模型的研究

摘要

探索更多视频

此视频中的章节

小鼠胰岛的分离和包膜下肾移植的方法

补肾胶囊对糖尿病小鼠的胰岛移植

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

应用改良袖套技术的小鼠胰腺移植模型

毛里岛皮下白脂肪组织（ISWAT）移植模型

尽量减少小鼠肝内胰岛移植期间输注后门静脉出血

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

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