这项工作是支持的，部分由一个津贴从美国国立卫生研究院（RO1 HL098228）到CKB。

注：所有动物的程序批准了全国儿童医院实验动物管理和使用委员会。 1，嫁接制造<ol><li>通过在通风橱中加入100毫克P（LA / CL）在2毫升二氧杂环己烷使ε-己内酯和L-丙交酯共聚物P（LA / CL）的溶液。将解决方案在涡不断为1-1.5小时至完全溶解混匀。 </li><li>在此期间，除去聚乙醇酸片材（PGA），从冷冻库毡和切出几个5毫米x 8毫米的部分。也切断0.1-10微升移液器只是过滤器上方的一角。 </li><li>插入19号针头（1.5长度）成吸管尖的前端和包裹在PGA毡针周围采用微型镊子。 </li><li>小心地将毛毡的吸管头，其中所述管腔是比近侧部分直，用钝18号针头的前端，而19号针头插入它。 </li><li>移液器40微升P（CL / LA）的溶液成从顶部枪头。饱和PGA毡用的解决方案。然后用移液管分配器推气泡出来。 如果需要，重复此过程。 </li><li>在50毫升试管移植地点，并将其放置在-80℃下冷冻20分钟。确保针的头部朝下。 </li><li>传输管放入冻干机及真空24小时。请务必打开管盖有气流。 </li><li>取出移植物，并从针头中删除。剪下移植留下了约5 mm截面的两端，把他们放回针来保持其形状。保持在干燥器的移植。 </li><li>在生物安全罩O / N前细胞种植放置支架在紫外光下。 </li></ol> 2，骨髓单核细胞收获和隔离<ol><li>安乐死的小鼠用氯胺酮/赛拉嗪过量（氯胺酮，200 mg / kg和甲苯噻嗪，20毫克/千克）。 </li><li>除去骨（股骨的ð胫骨）从3老鼠10移植植入并将其放置在培养皿中与10毫升的RPMI。切使用注射器用25号针头进入一个新的培养皿中以3毫升的RPMI骨骼和冲洗骨髓的两端。采集骨髓和RPMI解决方案在一个15毫升管，并与另外2毫升的RPMI洗培养皿收集BM的其余部分。 </li><li>取样品（5-10微升），并使用自动细胞计数器或血球计数细胞。记录结果。 </li><li>把5毫升聚蔗糖在15毫升离心管中，加入骨髓和RPMI解决方案。添加解决方案非常平缓，以防止它与聚蔗糖混合。 </li><li>在24℃下离心528×g离心30分钟，“没有刹车” </li><li>拆下上部粉红色层。收集的中间透明层图1，这是在MNC层，并用PBS 1点01稀释。 </li><li>离心跨国公司稀溶液在528×g离心10分钟，在24°C。 </li><li>除去上清液和二琵琶用5ml PBS中沉淀。 </li><li>离心沉淀溶液在528×g离心10分钟，在24℃下</li><li>除去上清液。淡化沉淀的RPMI（约200微升）的适当的音量。 </li><li>取5-10微升样品，并使用自动细胞计数器或血球计数细胞。记录结果。重复该细胞计数一次，并计算平均细胞数。 </li><li>稀释细胞浓度为1,000,000 cells/10微升用RPMI。 </li></ol> 3。细胞种植<ol><li>预湿脚手架加入5μLRPMI肠腔5分钟，然后取出RPMI。 </li><li>从步骤2.12中添加10μl的骨髓来源的单核细胞在RPMI到支架内腔并等待10分钟，以使细胞附着在支架上。 </li><li>切割一个19号针头至1厘米长，并把针头插入支架的内腔，以保持支架的形状。将样品在一个24孔板中。&lt;/ LI&gt; <li>新增1,000微升RPMI到每口井和孵化O / N中的孵化器。 </li></ol> 4，移植物植入<ol><li>高压灭菌所有的外科手术工具手术前：1倍罚款剪刀，3微型镊子，微型2倍血管钳，1x夹紧应用镊子，1个微型针器，1个弹簧剪刀，1卷收器。 </li><li> 6-8周龄雌性C57BL / 6被用作组织工程血管移植受者。从笼中取出小鼠并称重，然后通过腹膜内注射麻醉与氯胺酮/赛拉嗪混合物（氯胺酮，100 mg / kg和甲苯噻嗪，10毫克/千克）腹部的右下象限。酮洛芬（5毫克/公斤，腹腔注射）被用作预麻醉止痛剂。 </li><li>通过故事捏检查镇静水平，然后夹腹毛。润滑眼睛用无菌眼药膏，然后将鼠标在垫背部靠着位置。消毒腹部与优碘和酒精垫。合作VER鼠标用无菌的悬垂性和唯一暴露在切口区域。 </li><li>使从剑突到耻骨上区域下方的中线剖腹手术切口，并插入一个自保持牵开器。在包裹蘸生理盐水纱布的肠子。说白了定义下腹主动脉和腔静脉。 </li><li>放置在近端与主动脉和腔静脉的远端侧上的两个微血管钳然后直截了当地从腔静脉断面腔静脉分离主动脉。如果有必要，结扎腹主动脉分支与上锥形针10-0单丝缝线。 </li><li>植入的下腔静脉插入移植物的近端和远端用无菌10-0缝合结束吻合。修剪嫁接，一般1-2毫米，依赖于鼠标的解剖结构。固定移植物与一个线圈在两个近端和远端，并开始与来自移植物的另一侧4-5拆线连续缝合。整理正面后，翻转夹具和移植到另一侧，并缝合移植物的背面。在植入，冲洗移植物与肝素溶液频繁，以防止急性血栓形成。 </li><li>取出近端钳和通过应用外用无菌可吸收止血剂控制出血。当出血完全停止后，取出远端夹具和控制出血的方法相同。通过嫁接确保血液流量。 </li><li>使用6-0黑色尼龙单丝缝合线与包括螺纹针关闭腹部肌肉和皮肤的两层。 </li><li>注射0.5毫升皮下注射生理盐水，然后将鼠标在一个变暖垫恢复笼，直到鼠标是完全移动。恢复后，返回鼠标到一个新的笼子纸床上用品。给予止痛药（布洛芬，30毫克/千克，生活饮用水）48小时。 </li></ol>

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<li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Late-term+results+of+tissue-engineered+vascular+grafts+in+humans%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Late-term results of tissue-engineered vascular grafts in humans.</a> The Journal of Thoracic and Cardiovascular Surgery. 139, 431-436 (2010).</li>
<li>Shin'oka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Midterm+clinical+result+of+tissue-engineered+vascular+autografts+seeded+with+autologous+bone+marrow+cells%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells.</a> The Journal of Thoracic and Cardiovascular Surgery. 129, 1330-1338 (2005).</li>
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</ol>

作者什么都没有透露。

TEVG的小鼠模型是研究细胞和分子neotissue形成和狭窄的发展机制的重要工具。引晶的BM-MNC中所示的移植物11的种子细胞的两个组织学和SEM图像。细胞接种效率是利用DNA测定7还示出。利用该模型系统中，我们发现，细胞接种降低TEVG狭窄，这是失败的主模式在我们的人体临床试验3的发展的发生率。将接种的细胞迅速从TEVG消失，这表明它们通过旁分泌机制8,12发挥它们的作用。它也表明，新血管形成是一种免疫介导的再生过程，而新血管源于从相邻血管壁沿着所述支架的腔表面的血管细胞（EC和SMC）的向内生长。此外，正常的巨噬细胞浸润的血管neotissue形成至关重要，但是当这个过程是过度它导致的TEVG 7,8狭窄的发展。这些结果表明这种小鼠模型与人类疾病的相关性。在显微外科技术可以适应其它血管应用，包括动脉移植物13和动脉到静脉瘘。 我们每年工作超过1000 IVC介入移植植入和死亡率小于1％。对于一个成功的移植物植入，也有提几个要点。 第一，可注射麻醉，优选进行此操作，因为它是要打开鼠标周围几次吻合术中。麻醉的效果通常持续约一个小时，并在整个手术时间对于一个熟练的微外科医生是约30-45分钟，它提供了足够的时间来完成手术。如果动物在手术中醒来，0.05-0.1毫升的麻醉鸡尾酒的可以注射intramuscularly。 第二，大多数小主动脉分支需要被连接，以防止过量的血液损失，同时分离出下腔静脉，并从腹部主动脉。还要记住不要伤到下腔静脉时，从主动脉分离。 第三，当接枝缝合，两侧的第2缝合线是整个过程中最重要的步骤。当它们不小心地放置，这是难以分开的下腔静脉的前面和后面的层。如果该层是不明确，有较高机会无意中缝合在一起。而缝合下腔静脉的移植，它不要过度伸展下腔静脉，可引起流泪是很重要的。另外，还要确保不切断太多IVC取代移植物的长度相同。一般IVC的约1至2毫米除去更换3〜5毫米接枝由于下腔静脉的纵向张力。如果下腔静脉的长度相同被除去，它使吻合口MOSIS极具挑战性。 第四，有显著应变应变变化就TEVG形成和狭窄的发展。例如，非种子选手C57BL / 6小鼠（野生型）呈狭窄的一种更高的速率比SCID / BG小鼠（免疫缺陷小鼠）8,12。此外，根据我们的经验，SCID / BG小鼠更容易在比较C57BL / 6小鼠进行操作，由于其更硬的下腔静脉壁14。对小鼠的新菌株操作时牢记这一点。 接枝制造，BM-MNC收获和细胞种植过程是直线前进，有的只是一对夫妇的过程中要弄清。首先，对于接枝的制造，这是很重要的是不要&#39;叠&#39;毡，同时使用18G的钝针推。我们已经尝试了不同的方法，以毛毡下推到的移液管尖端的远端。推移植用钝针显示最好的结局，到目前为止。内钝针的直径比毡缠绕在针的外径稍大一点。因此，较小的针可紧贴地滑进更大的钝针与毡可以随后被向下推的力相等的量都围绕毡，从而防止毛毯的聚拢的周围。我们以前观察到的成束移植具有较低的通畅2周。此外，以消除所有的泡沫这是至关重要的，因为气泡会造成支架表面和血液孔穿过孔植入后会泄漏。其次，当骨头分开，BM-MNC收获，一定要清洁骨骼尽可能使它们不包括太多的肌肉或头发。如果需要的话，则建议的密度离心过程之前，使用纸巾过滤器。 TEVG的这个模型运作良好的静脉循环，这是一个低压力和高流速的系统。然而，在动脉循环中，一个高压和高流量系统中，我们观察到的破裂带，由于其快速降解特性的PGA移植物的高利率。这意味着该接枝失去了它的结构强度之前neotissue获得足够的强度以抵抗动脉压。我们已经用聚乳酸（PLA）以前移植，但表现出的动脉瘤形成患病率较高。也需要超过2年才能完全降解PLA 在体内 ，这是超过小鼠的寿命。对于动脉循环移植的制造技术未来的应用程序，例如，静电移植是目前正在开发中。

先天性心脏缺陷是影响近8％的活产婴儿在美国的严重情况。约25％的婴儿患有先天性心脏缺陷或2.4每1000活产的，需要侵入性治疗在他们的生活1的第一年。最有效的治疗先天性心脏疾病是重建手术。不幸的是，从使用目前可用的血管导管引起的并发症是术后发病率和死亡率的最显著的原因。 为了解决这个问题，我们开发了第一个组织工程血管移植（TEVGs）的临床应用2。 TEVGs从播种与自体骨髓单个核细胞（BM-跨国公司），并作为植入静脉导管先天性心脏手术的可生物降解聚酯管构成。结果表明，优秀的通畅率在1-3年的随访，但狭窄的发生率显著<s高达&gt; 3,4。很明显，是需要更好地了解血管neotissue形成及相关TEVG狭窄的发展机制。为了更好地了解TEVGs的发展和狭窄发展的机制，一个绵羊模型建立5,6。在这个模型中，TEVGs成功转型为生活器皿并分别在这两个形态和原生矿脉的功能类似。这种使用了大量的动物模型是在提供辅助TEVGs的临床应用重要的临床前信息的一个很好的第一步。然而，血管neotissue形成用大动物模型TEVGs的细胞和分子机制的充分了解是由于由于缺乏物种特异的分子工具的血管细胞表型的分子特征局限性的限制。为了克服这些缺点，TEVGs小鼠模型是由原因的迅速发展在小鼠遗传学和其广泛的molecula的研制Ř表征具有缩短的时间刻度的额外优势。 鼠下腔静脉插入模型忠实扼要重述，发生在大动物和人类的新血管形成的过程，但在短得多的时间过程6-9。这里，对于使用可生物降解的支架小型接枝制造一个详细的协议中，BM-MNC收获并进行隔离，脚手架BM-MNC播种，并在小鼠模型中移植物植入进行了描述。

<table><tbody><tr><td>Polyglycolic acid (PGA) felt</td><td>Biomedical Structures</td><td></td><td>Custome ordered</td></tr><tr><td>Pipet tip, 0.1-10 &amp;mu;l&amp;nbsp;</td><td>Fisher Sientific</td><td>02-707-456</td><td></td></tr><tr><td>Lyophilizer&amp;nbsp;</td><td>Labconco</td><td>7070020</td><td></td></tr><tr><td>RPMI medium 1604</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Petri dish</td><td>BD</td><td>353003</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>15 cc tube&amp;nbsp;</td><td>BD</td><td>352096</td><td></td></tr><tr><td>Ficoll</td><td>Sigma</td><td>10831-100ml</td><td>Also called &#039;Histopaque&#039;</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Littauer bone cutter 4.5&quot; Straight</td><td>Roboz</td><td>RS-8480</td><td>For BM harvesting</td></tr><tr><td>Forceps 4.5&quot;</td><td>Roboz</td><td>RS-8120</td><td>For BM harvesting</td></tr><tr><td>Scissors 4.5&quot;</td><td>Roboz</td><td>RS-5912</td><td>For BM harvesting</td></tr><tr><td>Microscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>C57BL/6J (H-2b), Female</td><td>Jackson Laboratories</td><td>664</td><td>8-12 weeks</td></tr><tr><td>Ketamine hydrochloride injection</td><td>Hospira Inc.</td><td>NDC 0409-2053</td><td></td></tr><tr><td>Xylazine sterile solution</td><td>Akorn Inc.</td><td>NADA# 139-236</td><td></td></tr><tr><td>Ketoprofen</td><td>Fort Dodge Animal Health</td><td>NDC 0856-4396-01</td><td></td></tr><tr><td>Ibuprofen</td><td>PrecisionDose</td><td>NDC 68094-494-59</td><td></td></tr><tr><td>Heparin sodium</td><td>Sagent Pharmaceticals</td><td>NDC 25021-400</td><td></td></tr><tr><td>Saline solution (sterile 0.9% sodium chloride)</td><td>Hospira Inc.</td><td>NDC 0409-0138-22</td><td></td></tr><tr><td>0.9% Sodium chloride injection</td><td>Hospira Inc.</td><td>NDC 0409-4888-10</td><td></td></tr><tr><td>Petrolatum ophthalmic ointment</td><td>Dechra Veterinary Products</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Iodine prep pads</td><td>Triad Disposables, Inc.</td><td>NDC 50730-3201-1</td><td></td></tr><tr><td>Alcohol prep pads</td><td>McKesson Corp.</td><td>NDC 68599-5805-1</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Fisher Scientific</td><td>23-400-118</td><td></td></tr><tr><td>Fine scissor</td><td>FST</td><td>14028-10</td><td></td></tr><tr><td>Micro-adson forcep</td><td>FST</td><td>11018-12</td><td></td></tr><tr><td>Clamp applying forcep</td><td>FST</td><td>00072-14</td><td></td></tr><tr><td>S&amp;amp;T Vascular clamp</td><td>FST</td><td>00396-01</td><td></td></tr><tr><td>Spring scissors</td><td>FST</td><td>15008-08</td><td></td></tr><tr><td>Colibri retractors</td><td>FST</td><td>17000-04</td><td></td></tr><tr><td>Dumont #5 forcep</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Dumont #7 - fine forceps</td><td>FST</td><td>11274-20</td><td></td></tr><tr><td>Dumont #5/45 forceps</td><td>FST</td><td>11251-35</td><td></td></tr><tr><td>Tish needle holder/forceps</td><td>Micrins</td><td>MI1540</td><td></td></tr><tr><td>Black polyamide monofilament suture, 10-0</td><td>AROSurgical Instruments Corporation</td><td>TI638402</td><td>For suturing the graft</td></tr><tr><td>Black polyamide monofilament suture, 6-0</td><td>AROSurgical Instruments</td><td>SN-1956</td><td>For musculature and skin closure</td></tr><tr><td>Non-woven sponges</td><td>McKesson Corp.</td><td>94442000</td><td></td></tr><tr><td>Absorbable hemostat</td><td>Ethicon</td><td>1961</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>18 G 1.5 in, Needle</td><td>BD</td><td>305190</td><td></td></tr><tr><td>25 G 1 in, Needle</td><td>BD</td><td>305125</td><td></td></tr><tr><td>30 G 1 in, Needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-700</td><td></td></tr><tr><td>Warming pad</td><td>Gaymar</td><td>TP-22G</td><td></td></tr><tr><td>Trimmer</td><td>Wahl</td><td>9854-500</td><td></td></tr></tbody></table>

implantation of inferior vena cava interposition graft in mouse model

接种骨髓单个核细胞（骨髓细胞）生物可降解支架通常用于重建手术治疗先天性心脏畸形。长期的临床结果显示出优异的通畅率，但是，与狭窄的显著发病率。为了探讨血管neotissue形成的细胞和分子机制，防止狭窄发展组织工程血管移植（TEVGs），我们制定了移植物的小鼠模型大约1毫米的直径。首先，TEVGs被从聚乙醇酸无纺布制成可生物降解的管状支架组装毡网眼涂有ε-己内酯和L-丙交酯共聚物。的支架，然后放置于冷冻干燥机，抽真空24小时，并在干燥器中储存直到细胞接种。第二，骨髓是从供体小鼠中收集和单核细胞中分离出密度梯度离心。第三，约百万细胞接种于支架培养的O / N。最后，将种子支架，然后注入在C57BL / 6小鼠肾下腔静脉移植物插入。植入移植物表现出良好的通畅性（&gt; 90％），无血栓栓塞并发症或动脉瘤形成的证据。这种小鼠模型将有助于我们理解和量化neotissue形成的TEVG的细胞和分子机制。

的TEVG植入的示意图示于图1。骨髓被从供体小鼠收获并用密度离心的单核细胞中分离出，然后接种到生物可降解的支架。该种子支架培养的O / N和植入到受体小鼠为下腔静脉介入移植。 图2示出了PGA-P（CL / LA）支架材料的扫描电子显微镜。内部直径约为1毫米的壁厚为约0.17毫米。总孔隙度为78.5％，平均孔径分别为45.4±17.6微米。 插入到C57BL / 6小鼠的下腔静脉一TEVG的总图像如图3所示。右植入（A）及第2周植入后（B）之后。 （C）显示本机下腔静脉的比较严重的形象。显而易见的是接枝充满neotissue，然而，它仍保持了原有的移植物的形状。我们以前的结果表明，接枝总降解需要长达12周10。 的2周植入TEVG超声B模式和彩色多普勒图像图4A-B显示了植入移植物的通畅性。一个土生土长的下腔静脉图像加入进行比较（ 图4，C）。细胞种植提高了通畅的移植显著，通畅率在第2周的非种子选手，接种移植为65％和91％，分别为（P &lt;0.05，Fisher精确检验）。 图5显示了细胞浸润和在TEVG细胞外基质沉积2周植入后。染色用H＆E染色植移植显示整个支架材料（A）的移植和剩余物neotissue形成。牡鹿和Masson三色染色显示弹性蛋白沉积在中timal层（B）和胶原在内侧层（C）。观察整个内膜层（D）内皮细胞衬里和融合平滑肌细胞存在于整个TEVG，特别是低于欧盟衬（E），就表明了CD31和αSMA染色分别。通过植入后的第二个星期，巨噬细胞浸润明显，与F4/80染色（F）表示。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51632/51632fig1highres.jpg" src="/files/ftp_upload/51632/51632fig1.jpg" /> 图1原理图的TEVG植入骨髓单核细胞从供体鼠收获，接种到可降解支架，然后植入作为一种干预下腔静脉移植到受体小鼠。 薄页=“总是”&gt; <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51632/51632fig2highres.jpg" src="/files/ftp_upload/51632/51632fig2.jpg" /> 支架的图2。扫描电子显微镜图像。平均外径为1.45毫米，管腔直径为1.1mm和长度为3mm。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51632/51632fig3highres.jpg" src="/files/ftp_upload/51632/51632fig3.jpg" /> 图3。植入TEVG 2周，原生腔静脉后。 a）在紧接植入后，B）植入后2周，C）本地IVC进行比较。 fig4.jpg“/&gt; 图4。超声图像2周，原生IVC。 A）2个星期。C）下腔静脉本地人比较后，B型和B）彩色多普勒TEVG的形象。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51632/51632fig5highres.jpg" src="/files/ftp_upload/51632/51632fig5.jpg" /> 图5。代表2个星期图像术后TEVG。 A）H＆E（内腔），B），牡鹿（粉红色=弹性蛋白），C）趣的三色（蓝=胶原），D）CD31（棕色=内皮细胞），E）αSMA（棕色=平滑肌细胞） 和 F ）F4/80（棕色=巨噬细胞）。

Watch this Scientific Journal Video about Implantation of Inferior Vena Cava Interposition Graft in Mouse Model at JoVE.com

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

为了提高我们的细胞和分子neotissue形成的知识，最近被开发的TEVG小鼠模型。移植物植入在C57BL / 6小鼠肾下腔静脉移植物插入。该模型能达到类似的结果那些在我们的临床调查来实现，但在一个远缩短时间过程。

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

implantation-of-inferior-vena-cava-interposition-graft-in-mouse-model

Nanyang Technological University

Research

JoVE Journal

Medicine

11.8K 次观看.  Nationwide Children's Hospital. 为了提高我们的细胞和分子neotissue形成的知识，最近被开发的TEVG小鼠模型。移植物植入在C57BL / 6小鼠肾下腔静脉移植物插入。该模型能达到类似的结果那些在我们的临床调查来实现，但在一个远缩短时间过程。 

视频: Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

小鼠异位心脏移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

科研

生物学

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

医学

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

颈椎异位心脏移植小鼠

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

免疫与感染

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

静脉介入型号:研究旁路移植通畅一个合适的模型

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

利用血管袖带的小鼠异位宫颈心脏移植

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

通过 Cuff 技术将组织工程血管移植物植入小鼠颈动脉

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

生物工程

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

腹部小鼠心脏移植模型中的血液回路重建

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

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 ...