，MC和AVDP是支持的博士后研究员在佛兰德（IWT-佛兰德），通过科学和技术创新促进研究所的补助金。 JT持有的一部分的时间临床研究奖学金（KOOR，）UZ Leuven的。 DV是一个博士后研究员，鲁汶大学的资助，DBOF/10/062的支持。 MMdC是一个博士后研究员，从Conselho国家PesquisaËDesenvolvimento（CNPq）和伊拉斯谟的赠款支持。研究是由IWT-佛兰德，由欧共体授予DIMI的（LSHB-CT-2005-512146）和体内分子成像研究组（IMIR）的鲁汶大学。我们要感谢詹姆斯·M·威尔逊创办的宾夕法尼亚大学矢量核心，他们的AAV6.2包装质粒的腺相关病毒载体生产的一种礼物。

1。小鼠交配，以获得所需的妊娠阶段时间交配怀孕NMRI小鼠，使他们怀孕18天（E18）（总妊娠E19.5）的手术时间。前和手术后他们被安置在过滤器顶部笼，免费使用水和周星驰在正常的室温和正常的日光。 2。 （IT）胎儿气管内注射（图1） <ol><li>首先提交怀孕NMRI小鼠全身麻醉与异氟醚1.5％O 2的混合物在1.5 L / min的。的异氟醚的水平取决于年龄和应变的鼠标，但总体上应该是异氟醚的水平，使动物在麻醉状态，然后把怀孕在加热鼠标垫（37°C），以保持体温在手术治疗。 </li><li>整个手术过程是由两位外科医生。一个外科医生将进行解剖孕鼠和随后曝光的第Ë胎儿的气管内注射。第二个外科医生将执行胎儿气管内注射本身。用无菌工具和一个无菌技术进行的手术过程。 </li><li>用聚维酮碘消毒腹部和执行中位数剖腹手术，妊娠子宫暴露。 Exteriorize一个子宫角的时间和孕囊数一数。每一个胎儿角操作和选择的基础上外置的胎头在后续步骤中的最佳位置 。选择最合适的胎儿通过子宫壁的可视化。鼻子胎儿的头应该指向的外科医生将注入进气管暴露后胎儿的头和固定向后（如解释在随后的步骤2.5）。 </li><li>首先通过一个6-0的聚丙烯（普理灵） 荷包缝合线 ，直径约1厘米，通过子宫壁和胎膜（羊膜卷材e和顶叶卵黄囊）在该区域将被暴露后对胎儿的头。缝合保持固定的肩膀起在子宫内的胎儿。接下来，做一个切口在子宫内的钱袋约0.8厘米，用锋利的剪刀。 </li><li>轻轻挤压头部和颈部的胎儿通过子宫切开术。拉轻轻地绕在脖子上紧荷包缝合，并将其固定在微蚊式钳的位置。 胎儿的头部保持在过伸由910一个5-0的polyglactin的的两钳放入口周围的上颌骨（薇乔）缝线。 </li><li>立体变焦显微镜下（×的放大倍率） 胎儿的气管是一个垂直的颈部切口尖锐，钝性分离的可视化 。颈部区域中的切口是约5毫米长。这是一种肤浅的切口，使用钝性分离，达到气管的相关组织进一步解剖。 </li><l&gt;在直视下通过立体显微镜下， 注入的物质（ 例如，荧光珠或病毒载体）的总体积为30μl的进气管，使用50μl的汉密尔顿玻璃注射器用30 G尖锐的针。除去的针后，注入的液体的流出最小保证了正确的注射。切口没有注射后封闭，。 </li><li>要更换头在子宫内 ，轻轻压在鼻子上。颈部第一追溯到进入子宫，随后由磁头。的鼻子在最后。下一步，关闭子宫切开术收紧钱袋。然后，注入约0.5毫升生理溶液共注入羊膜腔防止羊水过少。要做到这一点，将针插入切口内的钱袋。接下来，关闭在针的周围缝合，之后，注入生理盐水。在最后一个步骤中，缩回的针，接着，通过完全关闭的pURSE字符串。 </li><li> 关闭经产妇腹壁（腹膜和内侧和外侧的肌肉层）和皮肤用5-0薇乔连续缝合在两个单独的层。此后，渗透到0.2％利多卡因切口手术后的疼痛缓解。建议使用丁丙诺啡（0.05 - 0.1毫克/千克SC或IP）手术后疼痛缓解。无论是预防性宫缩，也没有使用抗生素。这些老鼠都保存在一个加热垫（37°C），直到他们完全康复（约1小时）。 </li></ol> 3。胎儿羊膜腔内注射（IA） <ol><li>参见第2.1节</li><li>用聚维酮碘消毒腹部和执行中位数剖腹手术，妊娠子宫暴露。 Exteriorize一个子宫角的时间和孕囊数一数。 </li><li> IA注入可以对所有的胎儿，因为它需要更少的时间来执行。此外，胎儿的位置是不是临界CAL相比，气管内注射。由于子宫壁半透明结构，如头部，四肢和尾巴和胎盘位置，胎儿是很好的可见。 </li><li>使用与上述（2.7）相同的针和注射器， 注入 30μl的胎儿的嘴附近的物质。这是可以做到最容易之间的胎儿头部和前肢，或下肢和尾部之间。仅在注射之后进行仔细检查，针的位置，以确保没有胎儿结构是在注射时，在与尖端接触。 </li><li>子宫被重新定位在腹部和， 关闭腹壁和皮肤上述（2.9）。 </li></ol> 4。注入胎儿的评估和交叉培养<ol><li>杀了大坝，使用经批准的安乐死方法36小时后，胎儿的干预（E19.5）。无需额外的颈椎错位的麻醉将是最好的方法，其他一切形式的安乐死，包括麻醉（ 如异氟醚，苯巴比妥）麻醉，甚至死亡的胎儿，你在任何时候都希望避免的。提供操作对胎儿的剖腹产 。操作胎儿在子宫内确定他们的位置。双角子宫，卵巢年底开始在羊膜囊的编号。 </li><li>分娩后，使用以下标准来评估胎儿的可行性 :（1）心跳（2）粉红色的皮肤颜色（与紫绀型）及（3）自发存在。将履行这些标准在养母的产仔一日龄的幼崽只幼崽。根据经验我们知道，用微弱的心脏跳动，皮肤紫绀色和/或最小的自发运动的幼崽往往会无法生存，由于拒绝从养母的乱抛垃圾或经产妇食人。 </li><li>送货后发可见染料通过IT或IA注射（ 例如红色荧光分子），能够由该染料（粉红色的存在下，以保证正确的喷射从注入的红色fluospheres）在胸（通过直接注射到肺部后IT注射剂，或通过吸入到肺部后IA注射）或腹腔（通过摄入）。这可能是由于新生小鼠的事实，是半透明的，允许在体内评估肺和胃。 </li><li> 标记正确的注入与中国水墨皮下的尾巴基础之上的幼崽。将所有的幼崽，满足4.2中提到的要求，在养母的乱抛垃圾。 </li><li>为了保证最大的接受和操作幼崽的生存 ，离开不超过10只，总在养母的产仔（她自己的1日龄的幼崽，以及交叉抚育幼崽）。在放置操作上的小狗S养母的乱抛垃圾，包括他们在床上用品材料的养母，她的粪便和尿液含有任何不熟悉的气味来掩盖。 </li><li>将笼在一个安静的环境 ，不要打扰的垃圾至少12小时（过夜）。 </li><li>次日，仔细评估的存活率促进通过计算所有的幼崽，使养母的幼崽之间的区别和操作上的幼崽（标有中国水墨和平均小于其他幼崽）。最大限度地减少操作时间，以避免这些幼​​崽的母亲的排斥和避免低温。在一般情况下，如果仔兔没有任何牛奶在他们的胃（这由于新生幼崽半透明性质是可见的），预后非常差进一步的这些动物的生存。 </li></ol> 5。代表性的成果 总体方案中描述的实验FIGURE 2。 气管内注射的最佳音量的测定为IT注射，以确定最佳的体积，我们凭经验选择了不同的范围从10的卷，20〜30微升（= 3/volume）。为了便于检测，我们选择了，注入红色荧光分子（fluospheres，分子探针，莱顿，荷兰）尺寸100纳米。 IT注射在E18老胎儿后，肺，24小时后收获，固定在4％多聚甲醛在4℃和6微米冰冻切片作了过夜。细胞核和肌动蛋白丝，用Hoechst 33258（Sigma-Aldrich公司，Bornem，比利时）和Alexa Fluor 488鬼笔环肽（Invitrogen公司，Merelbeke，比利时），分别为20分钟，在室温下进行染色。 Biorad公司光辉使用2100激光共聚焦显微镜LaserSharp2000.6软件卡尔·蔡司共聚焦图像。相对荧光（比红色到蓝色的荧光的代表fluospheres和核stainin克，分别使用ImageJ在线软件（图3））进行定量。虽然在胎儿手术的时间，仅注射后30微升检测回流，指示过量注入的流体，如通过测量相对荧光（方差分析量化30μl中，得到最高量的荧光信号在肺实质对于每一对采用Student的t检验结果，比较，* P &lt;0.05，*** P &lt;0.001）。 在胃肠道的肺组织和生物分布的定量评估fluospheres 接下来，我们要比较的效率为目标的胎鼠肺后IT与IA注射的。要做到这一点，，30微升fluospheres交付后的胎鼠肺IT或IA注射在E18怀孕的NMRI小鼠（N = 5％组）。 IT注射导致相比，在IA中的显着更高的交付fluospheres胎儿肺。路由（1.43±0.56和0.05±0.02的相对荧光（分别比fluospheres给Hoechst，学生t-检验，方差分析，*** P &lt;0.001）（图4的交流），未处理的对照胎儿被用于归一化的荧光背景信号。胃肠道是阳性的IT和IA注射的动物（图4天），由处理过的胎儿或阴性对照组动物（数据未示出）在其他组织中没有观察到红色荧光。 以下rAAV2/6.2介导的基因传递胎儿肺部的气管内和羊膜腔内注射的比较在比较这两种分娩方式注入荧光分子，我们要评估后，IT和IA注射用重组腺相关病毒载体病毒转导的效率和随后的基因表达。 rAAV2/6.2编码萤火虫荧光素酶（的波动）（3×10 10 GC /胎儿）的鸡β-肌动蛋白（CBA）启动子的控制下，在胎儿NMRI小鼠在注射组（n = 8）或IA组（n = 6）E18。剖腹产和培养，存活下来的幼崽后的非侵入性的生物发光成像（BLI）和，监测的涨落活动（光子/秒，P / S）的年龄在1周（图5）。 IT组的总光子通量，显着高于在IA组和阴性对照组（方差分析，比较每对学生的t检验，* P &lt;0.05）。在IA组的时间是平均BLI信号不显着高于在阴性对照组。 正确的和不正确的胎儿气管内注射的区别一个正确，一个incorrectI.T的区别。注射可以在几个层次上进行评估。手术的时间点，在注射过程中进入胎儿气管，没有阻力将会注意到当针然后e被定位在气管。然而，较高的电阻将会注意到时在气管空间注入。其次，剖宫产胎儿是半透明的，它是可能的看到的肺和其后的可见的染料的存在下（ 例如，中国墨，荧光分子）。最后一个选项，以评估正确的注射是通过光学成像和更具体的生物发光成像。 BLI是一个优雅的系统，以非侵入性的后续的报告基因的萤火虫荧光素酶的基因的表达，但在空间分辨率和解剖的信息是有限的。磁共振成像（MRI）提供高分辨率，断层图像包含详细的解剖信息。因此，我们调查相结合的BLI与MRI获得的叠加图像，它结合了一个可视化的更深层次的解剖结构（内脏）的表面BLI信号。我们的目的是要获得更详细的体内信息的本地化基因的表达能够区分正确的，从一个错误的IT注射。 结合BL-MR图像被收购一些动物注射IT rAAV2/6.2 CBA-涨落和CBA-LacZnls的的（3×10 10 GC /胎儿的每一个向量，n = 10）在一个星期的年龄（ 图6）。 BL成像显示，从颈部和胸部发出的信号。联合登记MRI BLI位于荧光素酶基因的表达在肺区域后，正确的注射（ 图6a），但在颈部和腹部区域后不正确的注入（ 图6b）。用X-gal染色的组织学分析证实了在体内共同登记。 气管内和羊膜腔内注射后的生存 <ol class="lalpha"><li> Fluorospheres 交付E18老NMRI胎儿的生存注入IT和IA 30μL100 nm红色fluorescen吨分子为100％，在这两个群体，并注入胎儿存活的数目定义为在24小时后收获的时间，胎儿的外科手术（ 表1）。 </li><li>腺相关病毒载体 交付的胎儿的生存注入IT或IA rAAV2/6.2，它被定义为胎儿存活剖宫产36h的胎后注射的时间点的数目的，分别为85.3％和86.3％，分别为（ 表1）。新生儿早期生存率为53.3％（IT）和74.5％（IA），并计算出关联的的幼崽活着1天数后培养的初始注入胎儿。要获得这些最终的存活率，我们优化了手术围手术期的程序使用（1）吸入异氟醚麻醉氯胺酮（75 mg / kg的IP），美托咪定的混合物，而不是管理（1 mg / kg的IP 协议 ） ，（2）的加热垫，以防止在手术过程中，（3）低温，立体变焦显微镜和（4）的运营商的越来越与外科手术经验的。 </li></ol><img alt="图1" src="/files/ftp_upload/4219/4219fig1.jpg" /> 图1。描绘在E18胎鼠气管内注射，在该图中对胎儿的IT注射的手术过程的主要步骤。在第一步骤中，一个子宫角形象化了。在下一步骤中，荷包缝合通过子宫壁的胎膜（羊膜和壁层卵黄囊）以上的区域中购买胎头将露出。接着，对头部和颈部的胎儿外置通过子宫切开术，胎头被保持在过伸5-0的polyglactin 910两钳钳口之间的缝合线。立体变焦显微镜下（×的放大倍率）胎儿的气管是由垂直脖子incis的可视化使用锋利，钝性分离离子。在最后一个步骤中，被注入的总体积为30μl的物质的立体放大显微镜直视下通过进气管。 <img alt="图2" src="/files/ftp_upload/4219/4219fig2.jpg" /> 图2。在实验的总体概述。 <img alt="图3" src="/files/ftp_upload/4219/4219fig3.jpg" /> 图3。气管内注射的最佳量的测定。为了确定最佳的IT注射量为10，20或30微升（N = 3/volume）红fluospheres E18老胎儿大小100 nm的管理和肺部收获24小时后来。用Hoechst 33258和Alexa Fluor 488鬼笔环肽染色，细胞核和肌动蛋白细丝。相对荧光（比红蓝色荧光，表示fluosphERE和核染色）进行了量化使用ImageJ的在线软件。平均值±SD，方差分析，每对使用学生t-检验比较，* P &lt;0.05，*** P &lt;0.001。 <img alt="图4" src="/files/ftp_upload/4219/4219fig4.jpg" /> 图4。定量评估fluospheres肺组织和生物分布的胃肠道，30微升红fluospheres的交付在（a）或（b）IA E18怀孕NMRI小鼠注射比较的效率目标的胎鼠肺胎鼠肺。未经处理的控制胎儿被用于正常化的荧光背景信号。核和肌动蛋白丝，用Hoechst 33258染色和Alexa Fluor 488鬼笔环肽（C）的相对荧光（红色比蓝色荧光，占fluosp继承人和核染色）进行了量化使用ImageJ的在线软件。（d）在胃肠道呈阳性的IT和IA注射的动物。平均值±标准差，学生的t检验，方差分析，*** P &lt;0.001。 <a href="http://www.jove.com/files/ftp_upload/4219/4219fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/4219/4219fig5.jpg" /> 图5。比较 BLI信号在1周后，（3×10 10 GC /胎儿CBA-涨落）与相应的总光子通量定量注射rAAV2/6.2 气管及羊膜腔内注射后，在胎儿肺rAAV2/6.2介导的基因传递。 。所有的动物进行扫描，由黑色的分区分开，以避免的光子散射到邻近的动物。伪规模描绘每秒的光子通量，每平方厘米每球面度器（p /秒/厘米2 / sr的）。测量中得到了4.3厘米2的矩形区域的景点。平均值±SD，方差分析，对于每对使用学生t-检验比较，* P &lt;0.05。图改编自卡隆等人 ，2010年。麦克米伦出 ​​版有限公司许可转载:[分子治疗（DOI:10.1038/mt.2010.153），版权（2010） <a href="http://www.jove.com/files/ftp_upload/4219/4219fig5large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图6" src="/files/ftp_upload/4219/4219fig6.jpg" /> 图6。区别胎儿肺部的rAAV2/6.2介导的基因传递正确的和不正确的胎儿气管内注射，结合BL-MR图像被收购一些动物注射IT与CBA-涨落和CBA-LacZnls的（3×10 rAAV2/6.2，GC /胎儿的每个vector）在年龄1周。 BL成像显示，从颈部和胸部发出的信号。联合登记MRI BLI位于荧光素酶基因的表达在肺后，正确的注射（一）的区域，但在颈部和腹部区域后不正确的喷射（二）。组织学分析证实了在体内共同注册。比例尺= 100微米。图改编自卡隆等人 ，2010年。 <a href="http://www.jove.com/files/ftp_upload/4219/4219fig6large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1"><tbody><tr><td> 注射物质 </td><td> 注射方法 </td><td> 求生存， 要在交货期 </td><td> 促进b的存活率 </td><td> 早期新生儿存活率 c </td></tr><tr><td> fluospheres </td><td> IT </td><td> 100（8/8）&lt;/ TD&gt; <td>不适用</td><td>不适用</td></tr><tr><td></td><td> IA </td><td> 100（5/5） </td><td>不适用</td><td>不适用</td></tr><tr><td> rAAV2/6.2 </td><td> IT </td><td> 85,3（64/75） </td><td> 62,5 </td><td> 53,3（七十五分之四十） </td></tr><tr><td></td><td> IA </td><td> 86,3（44/51） </td><td> 86,4 </td><td> 74,5（38/51） </td></tr></tbody></table> 表1中。 生存的交付， 即胎儿手术和剖腹产后，才培养。b只幼仔培育，如果他们是粉红色的，移动和呼吸正常的新生儿成活率表示胎儿气管内和羊膜腔内注射后的存活。作为一个功能的初始注入幼崽。缩写:IT气管内注射; IA羊膜腔内注射; na不适用。

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没有利益冲突的声明。

关键步骤<ul><li>小鼠品系，我们选择了与NMRI小鼠，因为他们有丰富的幼崽（平均窝产仔数14.4±1.8，自己的数据），以及容忍的干预，并具有良好的母体特征。 </li><li>通过子宫壁和胎膜配售的钱袋是关键的一步，因为你只是想揭露胎儿的头和肩膀，否则重新定位，几乎是不可能的，而不会造成创伤。 </li><li>胎儿的头向后伸展的最佳位置正上方的气管切开是必要的，以这样可以避免大血管（颈静脉）与气管平行。 </li><li>重要的是在视觉上跟随进入胎儿气管手术显微镜下，以避免不正确的注射针的插入。 </li><li>更换胎儿的头是至关重要的，因为你的子宫囊中，而不会造成创伤我想，以避免伤害胎儿的头，这会增加子宫内或出生后的死亡率。 </li><li>颈椎脱位，只有在被杀害的母亲剖腹产的时间点，而不是最初的CO 2窒息，因为这会产生负面影响胎儿的生存能力。 </li><li>交叉培养，以提高存活率的治疗经营的胎儿经阴道自然分娩的胎儿，才酿成了一个生存率为18.6±16.9％，去年同期为62±14％，非注射的幼崽，在我们的手中。 </li></ul>限制<ul><li>胎儿的IT注射耗费更多的时间来进行比较，IA注射。根据外科医生的经验，可以每2-4个胎儿孕鼠操作，以避免母鼠麻醉后一个多小时。 </li><li>一个30微升的量是最大的胎鼠气管注入。虽然有些泄漏可以检测国际家具展ediately注射后，注射后的30μl的荧光分子的荧光信号在肺中是最高的，如在图3中示出。 </li><li>最终的IT注射后的存活率低于后的IA注射（53.3％和74.5％），但主要是由于增加亏损后交叉培养。早期的成活率传递路线（85.3％和86.3％），胎儿外科手术的耐受性良好。 </li></ul>可能的修改和故障排除<ul><li>这可能是在更早的时间点，比E18可以进行胎儿IT注射。在较早的时间点执行胎儿手术胎儿的免疫系统可能是有利的，因为可能会对不太成熟，从而促进免疫耐受对病毒载体，治疗性蛋白质等，此外，扩大干细胞和祖细胞，可能会对更容易访问。针对这些细胞可能会引起到永久的遗传改正。但是，执行在较早的时间点可能会增加胎儿手术的发育畸变，其中有被监视的风险。 </li><li>手术后的护理幼崽接受注射胎儿，可以优化，更由于同类相食的动物他们在寄养巢后，以减少损失 。改变的小鼠品系的养母可能有所帮助。我们选择NMRI他们的好母亲的素质，但其他小鼠品系，如瑞士小鼠可能会​​表现得更好，为促进的目的。 </li></ul>未来的应用<ul><li>可用于单基因的致命疾病，如囊性纤维化，缺乏表面活性剂，α-1-抗胰蛋白酶缺乏胎儿基因治疗中的新颖的外科手术，针对由IT注射胎儿小鼠肺。产前治疗将是有益的，因为在这些情况下，治疗开始发病前的疾病，并且可以防止不可逆的损坏。此外，如果可以有针对性的干细胞或祖细胞，理论上可以以下方式获得终身的校正，因为这些细胞会不断提供后代表达缺陷蛋白。 </li><li>除了 ​​遗传性疾病，需要终身校正，胎儿的干预措施，旨在为一过性的治疗效果，将使用在欠发达的肺部需要临时基因表达的表面活性剂，血管内皮生长因子（肺成熟和新生血管），研究可能的治疗方案，为早产儿，或抗氧化剂蛋白， 如超氧化物歧化酶。 </li><li>用于产生疾病模型，此过程可进一步被用来提供在子宫内的化合物或毒素。例如，脂多糖处理，模拟宫内感染，可在子宫内干扰胎儿的肺发育，导致降低POS由于持续的慢性炎症和结构异常19 tnatal肺功能。 </li></ul>相对于现有的方法的技术意义<ul><li>由于胎儿呼吸道的特定靶向 ， 较高的转导效率 ，气道和肺泡注射病毒载体后，可以得到比现有的IA注入法。当病毒载体滴度限制的（ 例如慢病毒载体），将阻止它注射稀释的注入矢量入羊水，最大限度地量的载体颗粒在肺部。 </li><li>避免稀释后胎儿娩出也是有利的其他生物活性剂，如重组蛋白或干细胞的成本可以降低由于IT注射相比，IA注射所需的生理活性物质的量的小。 </li><li>胎儿IT我njection是独立的胎儿呼吸运动，开始出现在E14，但都是可变的动物个体之间的20。这样就产生了一种变异的特定吸收之间的IA注入胎儿，这可以降低由IT注射。 </li></ul>

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> NMRI小鼠</td><td>让维耶，乐圣岛，法国Genest </td><td></td><td></td></tr><tr><td>异氟醚</td><td> Isoba，英特威/先灵葆雅动物保健，米尔顿凯恩斯，英国</td><td></td><td></td></tr><tr><td>普理灵6-0 </td><td>爱惜，比利时，格罗特北臼阿登</td><td></td><td></td></tr><tr><td>薇乔5-0 </td><td>爱惜，比利时，格罗特北臼阿登</td><td></td><td></td></tr><tr><td> 50微升的汉密尔顿玻璃注射器，型号1710.5 TLLX SYR </td><td>汉密尔顿，雷诺，内华达州，美国</td><td> 5495-20 </td><td></td></tr><tr><td> 30G针</td><td>汉密尔顿，雷诺，内华达州，美国</td><td> 7762-03 </td><td></td></tr><tr><td> 2％利多卡因</td><td>阿斯利康（AstraZeneca），荷兰Zoetermeer </td><td></td><td></td></tr></tbody></table>

a novel surgical approach for intratracheal administration of bioactive agents in a fetal mouse model

产前肺部给药的细胞，基因或药物制剂可以提供各种遗传性和获得性疾病的新的治疗策略的基础。除了从先天性或继承异常要求交付的基因，几个非继承围产期条件，其中短期基因表达或药理干预是足以达到治疗效果的长期表达，被认为是作为潜在的未来的适应症种方法。候选短期产前治疗疾病的应用可能是的暂时性新生儿缺陷的表面活性蛋白B引起新生儿呼吸窘迫综合征1,2或高氧损伤的新生儿肺3。永久的治疗矫正的候选疾病囊性纤维化（CF）4，表面活性剂的不足5和α1-抗胰蛋白酶缺乏症的遗传变异。 &lt;P类="j​​ove_content"&gt;一般情况下，产前基因治疗的一个重要优势是能够开始在开发的早期治疗干预，甚至是在病人的临床表现之前，从而防止个人造成不可弥补的损害。此外，胎儿各器官的细胞增殖率增加相比，成人的器官，它可以让一个更有效的基因或干细胞进入胎儿体内转移。此外， 在子宫内的基因传递的个体的免疫系统时，进行不完全成熟。因此，异源细胞的移植或补充的非功能性的或缺失的蛋白质与一个正确的版本应该不会引起免疫致敏的细胞，载体或转基因产物，它最近已被证明是细胞和基因疗法的情况下，7 。 在本研究中，我们调查的潜在直接定位在小鼠胎儿的气管米ODEL。本程序是在使用在大型动物模型中，如兔，羊8，即使在临床的设置9，但迄今尚未在小鼠模型中之前进行。在研究潜在的胎儿基因治疗遗传性疾病，如CF，作为第一个证明了的概念，因为不同的转基因小鼠品系中，有据可查的胚胎发育和胎儿的发育，不那么严格的广泛使用的小鼠模型是非常有用的伦理规范，妊娠期短及大窝产仔数。 已经描述了不同的访问路线针对的胎儿鼠肺，包括羊膜腔内注射10-12，（超声引导）肺内注射13,14和静脉内给药到卵黄囊船只15,16或脐静脉17。我们的新型外科手术使研究人员能够直接注射剂的选择，允许进入胎儿小鼠气管为更有效地提供比现有技术18呼吸道。

Watch this Scientific Journal Video about A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model at JoVE.com

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

我们开发了一种新的手术方法气管内给药的生物活性剂的胎鼠。运送路线是更有效地针对胎儿比常用的羊膜腔内注射的小鼠肺组织。这个过程至今没有在小鼠模型中被描述。

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

a-novel-surgical-approach-for-intratracheal-administration-bioactive

Nanyang Technological University

Research

JoVE Journal

Medicine

16.4K 次观看.  KU Leuven. 我们开发了一种新的手术方法气管内给药的生物活性剂的胎鼠。运送路线是更有效地针对胎儿比常用的羊膜腔内注射的小鼠肺组织。这个过程至今没有在小鼠模型中被描述。

视频: A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

科研

医学

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

手术应激探索术后免疫抑制的机制和评估新围手术期免疫疗法的小鼠肿瘤模型

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型:在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

对大脑中动脉闭塞再灌注损伤诱发中风小鼠的手术方法

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or intraosseous routes, but in many patients, particularly in the emergency or critical care settings, these routes are often unavailable or time-consuming to access. A novel device is now available that offers an easy route for administration of medications or fluids via rectal mucosal absorption (also referred to as proctoclysis in the case of fluid administration and subsequent absorption). Although originally intended for the palliative care market, the utility of this device in the emergency setting has recently been described. Specifically, reports of patients being treated for dehydration, alcohol withdrawal, vomiting, fever, myocardial infarction, hyperthyroidism, and cardiac arrest have shown success with administration of a wide variety of medications or fluids (including water, aspirin, lorazepam, ondansetron, acetaminophen, methimazole, and buspirone). Device placement is straightforward, and based on the observation of expected effects from the medication administrations, absorption is rapid. The rapidity of absorption kinetics are further demonstrated in a recent report of the measurement of phenobarbital pharmacokinetics. We describe here the placement and use of this device, and demonstrate methods of pharmacokinetic measurements of medications administered by this method.

This study presents a novel device that offers an easy route to quickly provide medications and fluids in patients with limited or difficult IV access. This small-diameter device is placed in the distal third of the rectum, allowing for ongoing medication and fluids administration.

应急方案和设置药物和液体管理的新方法

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or ...

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. Critically ill patients require intensive medical support in order to survive. While the majority of patients will recover within a few days, about a quarter of the patients need prolonged intensive care and are at high risk of dying from non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness, and endocrine and metabolic changes, of which the pathogenesis is currently incompletely understood. The most widely used animal model in critical care research is the cecal ligation and puncture model to induce sepsis. This is a very reproducible model, with acute inflammatory and hemodynamic changes similar to human sepsis, which is designed to study the acute phase of critical illness. However, this model is hallmarked by a high lethality, which is different from the clinical human situation, and is not developed to study the prolonged phase of critical illness. Therefore, we adapted the technique by placing a central venous catheter in the jugular vein allowing us to administer clinically relevant supportive care, to better mimic the human clinical situation of critical illness. This mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a relevant model of the acute and prolonged phase of critical illness.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.

在重大疾病的小鼠模型中使用中心静脉导管的流体，药物和营养管理

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

小鼠体外循环模型: 一种新方法

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

应用新型磁吻合技术 Suprahepatic 腔静脉重建大鼠原位肝移植模型的研究

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...