这项工作是由美国国立卫生研究院资助R01 HL109420 WM和AHA 9320033N LY支持。

鼠标异体同基因移植模型所有的动物研究机构动物护理和使用委员会耶鲁大学批准。同种异体移植模型，使用一个终端到男，4-5周龄WT（C57BL/6J）或IFN-γRKO的小鼠胸主动脉段，插入女收件人，腹主动脉8-12周龄WT高端的显微外科吻合技术（有关详细信息，请参阅下）。 8-12周龄IFN-γR1-KO对于同基因移植模型，男，4-5周龄WT小鼠胸主动脉段插入到腹主动脉的男性，使用一个终端到终端的显微外科吻合技术。术后1周，动物接种IV与IFN-γAd5.CMV鼠或Ad5.CMV LacZ基因（QBIOGENE联系）1×10 9 PFU。血清小鼠IFN-γ水平测定ELISA（eBioscience公司）在1和5周后腺病毒管理。某些动物的BrdU（Sigma-Aldrich公司）在100毫克/千克SC 2周前牺牲。 终端到终端的显微外科吻合技术（这部分将采取视频）: 1。捐助者的程序<ol><li>麻醉供体小鼠腹腔注射氯胺酮（50毫克/公斤）和甲苯噻嗪（10毫克/千克）。 </li><li>确保有足够的麻醉后，在手术显微镜下将供体小鼠X8-30的放大倍率在仰卧位托盘上，并使用碘消毒片，酒精消毒片胸壁准备。 </li><li>切开通过前胸壁的肋骨和膈肌，暴露心脏。 </li><li>打开右心房进入左心室，注入10毫升的肝素（100 U / ml）的生理盐水冲洗小鼠用10毫升的注射器，25号针头。 </li><li>切除的心脏和肺部露出的整个长度胸主动脉。 </li><li>仔细解剖胸主动脉，以尽量减少直接创伤的whILË识别，结扎并用11-0附近的主动脉缝合木材划分分支机构。 </li><li>一旦胸主动脉周围组织，切除整个胸主动脉移植。 </li><li>肝素化（100 U / ml）的生理盐水溶液冲洗供体主动脉的切断端，并将其存储在冰上（最多6个小时），直到移植在同一溶液中。 </li></ol> 2。收件人程序<ol><li>同IP酮洛芬（5毫克/公斤）注入受体小鼠的镇痛，并，用ip氯胺酮（50毫克/千克）和甲苯噻嗪（10毫克/公斤）小鼠thenanesthetize。受体小鼠深深60-90分钟，持续时间在10分钟内麻醉。手术过程中，麻醉水平评估每隔15分钟用钳子捏腹前壁或脚。如果动物在手术过程中随时有害刺激的反应，追加剂量氯胺酮和甲苯噻嗪（1/4或1/3原剂量）会由SC或IM </li><li>确保有足够的麻醉后，腹前壁使用电动剃须刀剃掉皮草，清洁皮肤，使用碘消毒片，酒精片，使用眼药膏遮住眼睛。在手术过程中，将用于下列无菌技术操作，其中包括10％的漂白剂清洗运作表，戴无菌手套，使用无菌显微外科工具（蒸汽消毒仪器的操作开始时，再热玻璃珠灭菌多个操作间等）的仪器。 </li><li>将受体小鼠在手术显微镜下的放大倍率X8-30上的托盘在仰卧位。 </li><li>从剑突至耻骨中线切开腹壁，并波及腹壁除了使用微拉钩暴露腹腔。 </li><li>缩回的肠子优，用纱布包好，用生理盐水浸湿。受体小鼠变得损失HEA冷却t透外向的肠子。重要的是要保持动物的温暖。然而，加热板在手术过程中不使用适度低温有利于在防止脊髓损伤在闭塞主动脉的血流量，以及有小鼠体内不存在血流量的下半部分的传热效率。 </li><li>生殖器官的下部移动，用生理盐水溶液湿润的纱布包裹受体小鼠的腹部区域，并用它来缩回直肠腹部右侧。暴露的组织在移植过程中定期用盐水灌溉。 </li><li>解剖说穿了一段腹主动脉之间的肾血管主动脉分叉近端和远端仔细分离下腔静脉（IVC）。 </li><li>识别并结扎所有源于这个段腹主动脉，用11-0尼龙单丝缝合的小树枝。 </li><li>交叉夹紧的该隔离段使用bdominal主动脉血管钳近似5毫米，两端各​​一个。 </li><li>横切腹主动脉之间使用的是锋利的微型剪刀夹子创建吻合网站。 </li><li>切除从一个切断端的横断收件人主动脉腹主动脉的一小部分（不超过0.4毫米的长度），以适应供体主动脉移植物。 </li><li>冲洗使用肝素生理盐水溶液，以除去残留的血液的夹具之间的主动脉段。夹具和捐助主动脉移植的主动脉段之间的定期灌溉肝素（100 U / ml）的生理盐水溶液（40毫升/公斤）期间吻合。 </li><li>断面捐助主动脉两侧用锋利的微型剪刀，创建一个段嫁接移植长度为2.5毫米。 </li><li>原位位置，吻合移植供体的近端和远端分别到收件人的近端和远端腹主动脉切端，将捐助主动脉移植，最终吨邻图案，用11-0尼龙单丝缝合。 </li><li>对于连续的缝合线，将在3和9点钟位置首先住宿缝线。两个住缝线之间每侧移植，吻合两切边缘采用连续缝合3-4针，缝线连续缝合，留一个双结绑。由于封闭的两个层是连续的，裂开的风险增加。捐助主动脉移植应在适当的长度以连接收件人的近端和远端腹主动脉切端。 </li><li>间断缝合，构造四个吻合在3和9点钟位置两侧的接枝首先，3-4针迹之间的住宿缝线在移植物的每一侧的两个切割边缘吻合。 </li><li>松开钳远端允许逆行流入移植，确定出血部位，并在3分钟内作出额外的针。 </li><li>确定满意的止血后，松开近端钳。 </li><li>确认移植通畅大力脉动在接枝和本机腹主动脉的近端邻近节段的存在，特别是在远端邻近节段的腹主动脉和肠系膜下动脉。考虑，如果脉动的血流恢复后，在几分钟之内减少血栓形成的吻合口网站。 </li><li>返回肠道进入腹腔。 </li><li>缝合关闭腹壁肌肉层和皮肤层用5-0尼龙缝合线，分别。 </li><li>受体小鼠放置到一个干净的笼子一个加热垫的顶部，并等待1-2小时的老鼠，恢复知觉，并从麻醉中恢复。在恢复过程中，重要的是要保持动物足够的温暖。保持温暖和干燥的老鼠笼前动物从麻醉中唤醒。 </li><li>后肢运动功能评估后，老鼠恢复，并再次在第二日。一个成功的移植手术将证实我F无后肢功能障碍的受体小鼠中观察到第二天。 </li><li>管理与酮洛芬在受体小鼠的饮用水（5毫克/公斤/天= 27微克/毫升饮用水中）48小时。如果有任何的受体小鼠患上术后止痛药，疼痛缓解的新郎，揉成异常姿势，等失去流动性，失败证明了，他们将被实施安乐死。动物将被安乐死在预定结束点后1-60天。 </li></ol>嫁接分析采购同基因移植模型中在第4周和移植物动脉移植的同种异体移植后6周可操作（病毒感染后第5周），并分析通过标准组织学技术弹性van Gieson染色（EVG）染色法，苏木精和伊红（HE）染色和免疫染色。照片拍摄使用免疫荧光显微镜系统（蔡司）。细胞计数的细胞核阳性表达包围执行在高倍率下，平均5横截面为每个移植。接枝面积测量管腔内的内皮细胞，内膜（内皮细胞和内弹力层之间，IEL），媒体（IEL和外弹性膜之间，鳗鱼），壁厚（之间的内皮细胞和外弹性膜） 5串行交叉路段，相距150微米每个移植整个容器（EEL内）进行计算，采用计算机辅助的图像分析和NIH图像1.60（ <a href="http://rsbweb.nih.gov/nih-image/" target="_blank">http://rsbweb.nih.gov/nih-image</a> ）。 统计分析所有数据均以平均值±SEM。双尾配对t检验和双向方差分析进行使用的Prism软件程序（GraphPad软件的）。以P &lt;0.05的差异被认为是表明统计学意义。

<ol>
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专注于鼠标GA模型所描述的协议。该程序可以被应用到其他接枝移植模型。这些模型包括人性化的异种移植（ 即人类冠状动脉节段插入到免疫缺陷小鼠的肾主动脉红外线），急性排斥反应的鼠标GA模型（ 即鼠标从一个到另一个遗传应变收件人遗传应变主动脉）。我们描述的小鼠模型更接近人类的遗传病变。第一种模式涉及从雄性小鼠的血管段插入到相同的近交系（C57BL/6J）一名女收?...

接枝arteriosclerois的（GA），也称为移植物血管病变，是一个在移植器官的特点是弥漫性，圆周狭窄整个移植血管树7个月至数年发展的病理损害。早期阶段可能会导致偏心和焦距狭窄的动脉更明显，从而更紧密地类似看出，在传统的动脉粥样硬化狭窄。移植物血管内膜膨胀的结果，由于宿主的T细胞和巨噬细胞的浸润，特别是积累的细胞外基质和平滑肌细胞样细胞的管腔丢失源于接枝，主机或两个5，13，19，是不充分的补偿出境船舶重塑。临床上最显着的病变是心脏移植，心外膜和心肌内冠状动脉。最终，乔治亚州的冠状动脉将导致缺血性心脏衰竭。 GA是心脏GR年底的主要原因船尾损失。停在狭窄的GA缝合线，强烈暗示其发病移植同种抗原主机响应和领导分类GA作为一种慢性排斥反应3。然而，其他形式的动脉损伤可能增加GA的风险，可以通过增加净负担受伤或通过加强和/或调节同种免疫反应。接枝动脉的内皮细胞（EC）的衣料是保存在人类遗传算法和最表面的内膜的区域的相邻EC衣料宿主来源的IFN-γ产生的T细胞和巨噬细胞11，在最严重的渗透是在网站上一些患者供体特异性自身抗体，结合移植EC 16，但船只的发展是与GA表明几乎没有证据表明纤维素样坏死为特征的急性抗体介导的排斥11。 GA发病的最重要组成部分，是内膜平滑肌细胞内增殖，如果这个过程中可能会被逮捕，，GA是不可能的进步。我们组以前的工作表明，人类冠状动​​脉节段的内膜，插入红外线肾免疫缺陷小鼠主动脉扩大继转移的人类T细胞异体动脉捐助的，并且这个过程中和人干扰素可以抑制γ18。此外，外源人IFN-γ的可能导致内膜（内侧）在没有人类T细胞15，17在这些动脉移植物的血管平滑肌细胞（VSMC）增殖。 （这是重要的要注意，人类和小鼠IFN-γ不跨越物种，排除鼠标在这个实验系统主机的间接影响。）这些人性化的小鼠模型，谈到人类Ţ细胞/血管细胞相互作用的利益和内膜病变主要是由人类细胞（ 即移植派生）已观察到的临床标本中，但它们完全不重述临床情况，因为它们忽略宿主巨噬细胞和可能的其他类型的细胞的作用，在临床移植病变。在此过程中常规的小鼠模型理论上可以解决这个问题，有一个完整的宿主的免疫系统，并提供附加的优点，允许功率基因小鼠的方法被应用到GA补充人源化模型的限制。两个最广泛使用的小鼠模型涉及异位心脏移植和原位动脉移植1。异位心脏移植的动脉病变的发展主要是由宿主细胞，骨髓起源可能，而人类心脏移植的动脉内膜细胞主要是移植原点5，13，19。这是一个显着的区别，使我们开发替代小鼠模型。介入的鼠标主动脉另一个鼠标应变收件人一株成是人类慢性排斥反应的模型更为有限，因为急性细胞介导的​​排斥反应，这种小鼠模型完全消除所有捐助源性血管内细胞移植两三个第19周。因此，随后的变化中看到插入血管段是单纯的响应宿主细胞重新填充脱细胞血管支架，创造一个的高人造的情况作为模型移植血管的变化发生在诊所的相关性有限。最近，我们已经开发了两个新的小鼠模型来规避这些问题21。第一种模式涉及从雄性小鼠的血管段插入到相同的近交系（C57BL/6J）一名女收件人。第二个模型涉及到主机鼠标缺乏REC一脉相承和性别从一个野生型C57BL/6J鼠标的捐助中插入动脉段eptor IFN-γ（IFN-γRKO的），其次是管理的鼠标干扰素-γ（交付通过腺病毒载体感染的小鼠肝脏，在这里，我们将详细介绍协议的优势我们的鼠标GA模型。

<table border="1" >
<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>C57BL/6J (H-2b)</td>
 <td>Jackson Laboratories 
 (Bar Harbor, ME)</td>
 <td>000664</td>
 <td>Donor (5 weeks) 
 Recipient (8-12weeks)</td>
 </tr>
 <tr>
 <td>Ketamine Hydrochloride Injection </td>
 <td>Hospira Inc.</td>
 <td>NDC 0409-2053</td>
 <td>Storage Solution(50 mg/ml) 
 Working Solution(5 mg/ml)</td>
 </tr>
 <tr>
 <td> Xylazine Sterile Solution</td>
 <td>Lloyd Inc.</td>
 <td>NADA# 139-236</td>
 <td>Storage Solution(100 mg/ml) 
 Working Solution(1 mg/ml)</td>
 </tr>
 <tr>
 <td>Ketoprofen</td>
 <td>Fort Dodge Animal Health</td>
 <td>NDC 0856-4396-01</td>
 <td>Storage Solution(100 mg/ml) 
 Working Solution-oral 
 (0.027 mg/ml)</td>
 </tr>
 <tr>
 <td>Heparin Sodium</td>
 <td>Sagent Pharmaceticals</td>
 <td>NDC 25021-400</td>
 <td>Storage Solution(1000 U/ml) 
 Working Solution(100 U/ml)</td>
 </tr>
 <tr>
 <td>Saline solution (Sterile 0.9% Sodium Chloride)</td>
 <td>CareFusion</td>
 <td>AL4109</td>
 <td></td>
 </tr>
 <tr>
 <td>0.9% Sodium Chloride Injection</td>
 <td>Hospira Inc.</td>
 <td>NDC 0409-4888-10</td>
 <td>To prepare the anesthetic </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>Microscope</td>
 <td>Leica</td>
 <td>MZ95</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Scissors</td>
 <td>Roboz Surgical Instrument Co.</td>
 <td>RS-5693</td>
 <td></td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>F.S.T</td>
 <td>15009-08</td>
 <td>To transect the aorta of donor or recipient </td>
 </tr>
 <tr>
 <td>Extra Narrow Scissors</td>
 <td>F.S.T</td>
 <td>14088-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle Holder/Forceps</td>
 <td>MICRINS</td>
 <td>MI1542</td>
 <td>To hold the needle</td>
 </tr>
 <tr>
 <td>Fine Forceps</td>
 <td>F.S.T </td>
 <td>11254-20</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>F.S.T</td>
 <td>11251-35</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Pattern Forceps</td>
 <td>F.S.T</td>
 <td>11000-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>F.S.T </td>
 <td>13011-12</td>
 <td></td>
 </tr>
 <tr>
 <td>LANCASTER Eye Speculum</td>
 <td>Zepf Medical Instruments</td>
 <td>42-1209-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Vascular Clip</td>
 <td>Roboz Surgical Instrument Co.</td>
 <td>RS-6472</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Clip Applying Forceps With Lock</td>
 <td>Roboz Surgical Instrument Co.</td>
 <td>RS-5440</td>
 <td></td>
 </tr>
 <tr>
 <td>Black Polyamide Monofilament Suture</td>
 <td>AROSurgical Instruments Corporation</td>
 <td>Cat #T4A10Q07</td>
 <td>10-0 suture, Needle=70 microns</td>
 </tr>
 <tr>
 <td>Black Monofilament Nylon Suture</td>
 <td>Syneture 
 (Covidien)</td>
 <td>SN-1956</td>
 <td>6-0 suture</td>
 </tr>
 <tr>
 <td>Non-Woven Songes</td>
 <td>McKesson Corp.</td>
 <td>Reorder No. 94442000</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml Syringe</td>
 <td>BD</td>
 <td>REF 309659</td>
 <td></td>
 </tr>
 <tr>
 <td>3 ml Syringe</td>
 <td>BD</td>
 <td>REF 309657</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml Syringe</td>
 <td>BD</td>
 <td>REF 309604</td>
 <td></td>
 </tr>
 <tr>
 <td>18G 1 1/2, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305196</td>
 <td></td>
 </tr>
 <tr>
 <td>25G 7/8, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305124</td>
 <td></td>
 </tr>
 <tr>
 <td>27G 1/2, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305109</td>
 <td></td>
 </tr>
 <tr>
 <td>30G 1/2, 
 Hypodermic Needle</td>
 <td>BD</td>
 <td>REF 305106</td>
 <td></td>
 </tr>
 <tr>
 <td>Hearting Pad</td>
 <td>Sunbeam</td>
 <td>Z-1228-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Trimmer</td>
 <td>Wahl</td>
 <td>9854-500</td>
 <td></td>
 </tr>
<tr>
<td></td>
<td></td>
<td></td>
<td>Table 2. Specific reagents and equipment.</td>
</tr>
 </tbody>
</table>

mouse models for graft arteriosclerosis

接枝arteriosclerois的（GA），也称为移植物血管​​病变，是经过数月至数年在弥漫，整个移植血管树的圆周狭窄的移植器官的特点，开发一个病理病变。 GA发病的最重要组成部分是内膜平滑肌细胞内的增殖。当一个人冠状动脉段被插入到红外线的免疫缺陷小鼠的肾主动脉的内膜可以扩大在人类T细胞过继转移反应同种异体动脉供体或外源性的人IFN-γ中的人T细胞的缺乏。从一株成另一种小鼠品系收件人鼠标主动脉介入有限，因为人类的慢性排斥反应的模型，因为这种小鼠模型中的急性细胞介导的​​排斥反应，完全消除所有捐助源性血管内细胞移植两三个周。最近，我们已经开发了两种新的m乌斯模型来规避这些问题。第一种模式涉及从雄性小鼠的血管段插入到相同的近交系（C57BL/6J）一名女收件人。次要组织相容性抗原编码的Y染色体（本在男但不是女）的，随之而来的是足够了几个星期，懒惰保留供体来源的平滑肌细胞的排斥反应，移植物排斥在这种情况下，仅仅针对的。第二个模型涉及动脉段插​​入到主机从一个野生型C57BL/6J鼠标的捐助鼠标一脉相承和性别，缺乏干扰素受体-γ其次是管理的鼠标IFN-γ（通过感染的鼠标交付肝脏的腺病毒载体。没有在这种情况下，作为供体和受体的小鼠相同的应变和性别，但供体平滑肌细胞增殖中的细胞因子，而宿主来源的细胞，缺乏受体抑制这种细胞因子，反应迟钝。通过回交附加到容器中供体的基因变化，这两种模式可以用于特定基因的遗传进展的效果进行评估。在这里，我们描述了我们的鼠标GA模型的详细协议。

小鼠移植物动脉硬化（GA）模式 :在这种模式下，男性捐赠者主动脉移植到女性的收件人，使主机诱导同种异体反应性T细胞介导的免疫性反应表示对未成年y抗原（男性特有的次要组织相容性抗原HY）移植物12，反过来T细胞产生IFN-γ的驱动器的血管平滑肌细胞增殖20中观察到的其他同种异体移植模型2，6，8-10，14。从捐赠者男A段胸主动脉手术，插入女性收件人的...

Watch this Scientific Journal Video about Mouse Models for Graft Arteriosclerosis at JoVE.com

Mouse Models for Graft Arteriosclerosis

我们描述了协议我们的鼠标接枝arteriosclerois的的模型（GA）涉及鼠标容器段插入到相同的近交系的收件人。通过回交额外的遗传变为血管捐助，该模型可以评估特定基因遗传的影响。

嫁接动脉硬化的小鼠模型

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Research

JoVE Journal

Medicine

13.6K 次观看.  Yale University School of Medicine. 我们描述了协议我们的鼠标接枝arteriosclerois的的模型（GA）涉及鼠标容器段插入到相同的近交系的收件人。通过回交额外的遗传变为血管捐助，该模型可以评估特定基因遗传的影响。

视频: Mouse Models for Graft Arteriosclerosis

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

科研

医学

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI和PET心肌梗死小鼠模型

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

生产用锌指核酸载脂蛋白C-III淘汰赛兔

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

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. ...

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 ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

的同种免疫引起的血管性排斥反应和移植物动脉硬化小鼠模型

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

对小鼠淋巴瘤模型监测肿瘤进展和治疗效果生物发光基于肿瘤定量方法

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

皮肤去神经外科手术:一种用于皮肤病的小鼠模型测试要求的神经方法

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

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. ...