作者想感谢约翰马斯顿畜牧业专家和博士马可波罗A. Passini技术援助，在这个项目的早期阶段。这项工作是由资助的​​国立卫生研究院慢性淋巴细胞白血病（R01NS41584; R01HD054413）。

1。侧脑室注射<ol><li>第一步是准备注入股市的解决方案，这些解决方案是病毒载体，质粒DNA，药物，应在无菌条件下注入。 </li><li>混合0.05％的注射部位的可视化W / V台盼蓝的PBS所需滴度的病毒载体（5-7微升总）。 </li><li>质粒DNA溶液（5升微升总）包含的D -（+） - 葡萄糖20％（W / V）（1μL），台盼蓝（0.05％），PBS（1μL），质粒（约5〜G /〜L ）（2〜L）和2.5 kDa的线性PEI均聚物（150毫米）（1〜L）。 </li><li>通过1-2分钟，低温麻醉，固定的PND 2新生儿。 </li><li>使用此注射针微升校准灭菌的玻璃微管，是通过长管连接到一个3毫升3毫升汉密尔顿注射器。 </li><li>打破（锅铲）调整为2毫米渗透入颅的针头的针尖。斜中含有的微量注射液管将针头。仔细地拉动注射器的柱塞装入针注射液。 </li><li>摄取的解决方案后，脱离管注射器;拉柱塞进一步，然后再次连接到针。 </li><li>在右手拇指和食指之间保持的针头和地方注射器柱塞摸右手手掌的中指和无名指之间。 </li><li>抓紧背后的头部皮肤使用左手和地方在光纤光照亮相关的解剖结构，可作为指导使用固定的鼠标。 </li><li>针2毫米深，垂直插入颅骨表面，在一个位置约0.25毫米，横向的矢状缝和0.50-0.75毫米，喙新生儿冠状动脉缝合（ 图 1A）。然后，用手掌推柱塞右手非常缓慢和谨慎。监测破裂的船只或面部肿胀。 </li><li>取出针15秒后停止活塞运动，以防止回流。 </li><li>要恢复，保持5-10分钟的小鼠，在加热的容器中，直到恢复运动和一般的响应。 </li></ol> 2。通过时间/面部静脉的静脉注射<ol><li>被注入的解决方案应该是准备在1:100稀释，并辅以过滤绿色食用色素和无菌条件下注入。 </li><li>将一个小塑料露儿100μL玻璃汉密尔顿注射器年底。露儿，附加33计0.25英寸的注射针。确保所有部件连接牢固。 </li><li>被注入量装入注射器，确保在注射器注射入​​血管的空气，没有空气是致命的。它往往是有益的吸管要你们一块的封口膜注射量。这允许摄取到注射器的解决明确的可视化。 </li><li>一个极小的景象透射是用来方便地可视化的颞浅静脉，或在新生儿的面部静脉（图2A）。注射前，确保新生儿使用手术磁带的透射。纱布应该放在新生儿和破坏动物的皮肤，以防止磁带的磁带。安全的动物，在其一侧的透射，确保磁带并不妨碍呼吸。新生儿的脖子上，应轻轻打开，使面部静脉是很容易看到，贴在鼻子应该是稳定的头部。 </li><li>使用2.25X头带放大镜，以便更容易可视化，慢慢地插入静脉的针。使针头应保持可见皮下的静脉是很肤浅的。 </li><li>静脉缓慢注入溶液的体积。绿色染料允许注射容易可视化。 </li><li>前等待15秒，取出针有一个完全压下注射器的柱塞和驱逐剩余的解决方案，由于极其狭窄的针孔之间的滞后。 </li><li>取出针后，用纱布注射部位施加压力，直到出血停止。 </li><li>监测新生儿窘迫的迹象。正确的注射后，没有明显的窘迫应得到遵守。新生儿应给予恢复它返回到笼前约5分钟。 </li><li>针/注射器单位之间的注射用PBS冲洗针的血液会很快堵塞由于小口径。 </li></ol> 3。代表性的成果: 侧脑室注射:大脑可以在不同的时间点注射后收获和可视化，以证明成功的注射技术。如果不添加染料注射液，将很难​​检查中的准确性jection。适当注射脑室之一，将允许在注射侧大脑的台盼蓝分布，注射后（ 图 1B）约10-15分钟。一台盼蓝均匀分布在左，右大脑半球和嗅球将可见注射后约60分钟（ 图1C）。这是由于连接将允许传播到相邻的脑室蓝色染料的脑室。精确的注射，也可以由散布在约12小时内注射后（ 图1D）延髓中央椎管蓝色染料的可视化。不准确的注射可以区分蓝色缺乏大脑半球。在这种情况下，进针深度是不足够的注射液有可能分布在皮肤下。另一种可能性是，针穿透过深在大脑中，从而超越了脑室及脑室底下掏空的解决方案是传递。 静脉注射:准确，高效的时间/面部静脉注射，可以可视化紧随注射。由于注入的解决方案，绿色染料允许可视化解决方案的进入和流经面部静脉（ 图2A）。此外，改变皮肤颜色的鼠标将遵守以下注射。新生儿正常的粉红色转换为绿色，由于绿色食品染料注射液存在（ 图2B） 。观察新生儿的绿色变化的程度取决于所使用的染料浓度。注射，并在实践期间的简单的可视化，1:50稀释建议。然而，显着地改变肤色，结果往往是小狗被母亲拒绝，因此，去除的染料或一个显著减少的浓度应在实验程序考虑。一个不准确的注入将导致绿色染料的积累，在注射部位的皮肤下。注射不完整的，它发生时没有完全插入静脉内针开幕，在静脉中流动时，会出现一些解决方案和一些积聚在皮肤下。如果发生此种情况，应插入针入血管。 <img src="/files/ftp_upload/2968/2968fig1.jpg" alt="图1"/> 图1示范成功的ICV PDN2小鼠注射。 一个利用侧脑室注射入脑室注射部位）示意图B）从ICV的大脑注入左心室与台盼蓝的PBS小狗，合影留念注射后15分钟，C左心室）侧脑室脑注射台盼蓝的PBS小狗，合影留念注射后60分钟，D）延髓中央椎管在侧脑室注射用台盼蓝的PBS小狗左心室上，采取12小时注射后的照片。 <img src="/files/ftp_upload/2968/2968fig2.jpg" alt="图2"/> 图2示范成功的第四PDN2小鼠注射。静脉注射剂使用的面部静脉）示意图 B）小狗注射绿色染料第四的PBS相比，联合国注入littermate，照片立即注射后。

<ol>
	<li>Blanchette, M., Fortin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+in+the+treatment+of+brain+tumors.">Blood-brain barrier disruption in the treatment of brain tumors.</a> Methods Mol. Biol. 686, 447-463 (2011).</li><li>Foust, K. D., Kaspar, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+the+barrier+and+through+the+blood:+to+CNS+delivery+we+go.">Over the barrier and through the blood: to CNS delivery we go.</a> Cell Cycle. 24, 4017-4018 (2009).</li><li>Snyder, E. Y., Taylor, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cell+engraftment+corrects+lysosomal+storage+throughout+the+MPS+VII+mouse+brain.">Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.</a> Nature. 374, 367-370 (1995).</li><li>Passini, M. A., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+gene+delivery+and+structure-specific+patterns+of+expression+in+the+brain+after+intraventricular+injections+of+neonatal+mice+with+an+adeno-associated+virus+vector.">Widespread gene delivery and structure-specific patterns of expression in the brain after intraventricular injections of neonatal mice with an adeno-associated virus vector.</a> J. Virol. 24, 12382-12392 (2001).</li><li>Kienstra, K. A., Freysdottir, D., Gonzales, N. M., Hirschi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+neonatal+intravascular+injections:+modeling+newborn+disease.">Murine neonatal intravascular injections: modeling newborn disease.</a> J. Am. Assoc. Lab. Anim. Sci. 46, 50-54 (2007).</li><li>Sands, M. S., Barker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+intravenous+injection+in+neonatal+mice.">Percutaneous intravenous injection in neonatal mice.</a> Lab. Anim. Sci. 49, 328-330 (1999).</li><li>Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+the+spinal+muscular+atrophy+phenotype+in+a+mouse+model+by+early+postnatal+delivery+of+SMN.">Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.</a> Nat. Biotechnol. 28, 271-274 (2010).</li><li>Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Passini, M. A., Watson, D. J., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+the+mouse+brain+with+adeno-associated+virus.">Gene delivery to the mouse brain with adeno-associated virus.</a> Methods Mol. Biol. 246, 225-236 (2004).</li><li>Coady, T. H., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-splicing-mediated+improvement+in+a+severe+mouse+model+of+spinal+muscular+atrophy.">Trans-splicing-mediated improvement in a severe mouse model of spinal muscular atrophy.</a> J. Neurosci. 30, 126-130 (2010).</li><li>Baughan, T. D., Dickson, A., Osman, E. Y., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+bifunctional+RNAs+that+target+an+intronic+repressor+and+increase+SMN+levels+in+an+animal+model+of+spinal+muscular+atrophy.">Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy.</a> Hum. Mol. Genet. 18, 1600-1611 (2009).</li><li>Coady, T. H., Baughan, T. D., Shababi, M., Passini, M. A., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+single+vector+system+that+enhances+trans-splicing+of+SMN2+transcripts.">Development of a single vector system that enhances trans-splicing of SMN2 transcripts.</a> PLoS One. 3, e3468-e3468 (2008).</li><li>Dickson, A., Osman, E., Lorson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively-Acting+Bifunctional+RNA+Increases+Survival+Motor+Neuron+in+vitro+and+in+vivo.">Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron in vitro and in vivo.</a> Hum. Gene. Ther. 19, 1307-1315 .</li><li>Mattis, V. B., Ebert, A. D., Fosso, M. Y., Chang, C. W., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+a+read-through+inducing+compound,+TC007,+lessens+the+severity+of+a+spinal+muscular+atrophy+animal+model.">Delivery of a read-through inducing compound, TC007, lessens the severity of a spinal muscular atrophy animal model.</a> Hum. Mol. Genet. 18, 3906-3913 (2009).</li><li>Williams, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide-mediated+survival+of+motor+neuron+protein+expression+in+CNS+improves+phenotype+in+a+mouse+model+of+spinal+muscular+atrophy.">Oligonucleotide-mediated survival of motor neuron protein expression in CNS improves phenotype in a mouse model of spinal muscular atrophy.</a> J. Neurosci. 29, 7633-7638 (2009).</li><li>Passini, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-targeted+gene+therapy+improves+survival+and+motor+function+in+a+mouse+model+of+spinal+muscular+atrophy.">CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.</a> J. Clin. Invest. 120, 1253-1264 (2010).</li><li>Shababi, M., Glascock, J., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+SMN+Trans-Splicing+and+a+Neurotrophic+Factor+Increases+the+Life+Span+and+Body+Mass+in+a+Severe+Model+of+Spinal+Muscular+Atrophy.">Combination of SMN Trans-Splicing and a Neurotrophic Factor Increases the Life Span and Body Mass in a Severe Model of Spinal Muscular Atrophy.</a> Hum. Gene. Ther. 22, 1-10 (2010).</li></ol>

没有利益冲突的声明。

疾病的小鼠模型的研究往往需要药物或其他物质的管理，新生儿。在这段视频中，我们展示了一步一步程序，利用侧脑室（ICV）注射CNS涉及两种类型的注射战略目标的中枢神经系统，可用于:1）直接注射;或2）静脉注射针对时间/面部静脉。这些注射的时间是非常重要的。由于ICV注射写意，头骨必须相对可塑性。这种类型的注射是唯一可能通过的第一个星期的生活，变得越来越具有挑战性的和有害动物年龄的动物。然而，主要好处是，直接传递到中枢神经系统的实现，有效地绕过血脑屏障。然而，因此，如果调查人员正在开发中枢神经系统疗法，它可以分析，并不完全如血脑屏障通透性的药物性能进行了优化的化合物，化合物活性可测疾病相关的组织。在生命的最初几天的时间静脉使用已被​​证明是有价值的，因为这是一个相对非侵入性技术，一般静脉注射。在这一发展阶段，没有额外的静脉，如尾静脉在5生活的第二个星期的访问变得更加方便。这两种注射技术是安全的，可以执行与写意，小鼠注射后不久，没有任何有害的副作用中恢复。这两种技术适合于组的动物在很短的时间内快速注射。他们需要适当的可视化的目标区域，以确保成功的注射。这是所提供的透射和放大镜头带（ 见表1）静脉注射和侧脑室注射光纤光。 静脉注射的优势之一，是一个体积较大（100∞LL）可与注入〜90％的成功率 6 。同时学习四技术遇到的常见问题包括针错位导致皮下注射和/或不完整的注射的。这些问题都可以反驳，确保针头斜面是通过船只彻底螺纹。针插入船只远远不够，针头斜面完全是由船只包围。此外，船只是非常肤浅的，因而喷油器必须确保针头在血管和下方。要检查，如果针头在血管，针放置后应轻轻移动一边到另一边。如果针渗透的船只，船只将沿着针。如果船只不动，针未正确插入。执行此检查注射前，会增加成功的注射。新生儿提供AAV9向量最近已成功地利用电机使用在SMA 小鼠 7,8的神经元重组AAV载体转导的一个高比例。 在ICV注入的最重要的考虑因素，涉及到针尖的长度。笔尖必须足够长，这样它可以渗透在头骨至少2毫米。如果提示是不够长，针将无法有效地进入心室。另外，如果提示是太长，这阻碍了吸收被注入的解决方案，也创建了一个脆弱的一角，是很难通过头骨的正确引导。进针的角度，是重要的，确切的渗透入脑室，以确保它垂直走向颅骨矢状缝的一角。许多研究已利用侧脑室注射引入到病变的小鼠模型4,9-17中枢神经系统的化合物，药物，治疗的RNA，DNA质粒和病毒载体。然而，在侧脑室注射的限制因素是容量小的新生小鼠侧脑室可能不允许渗透所需的治疗药物量。因此，能否在很短的时间内使用一个较低的载体剂量，达到治疗的表达水平显得尤为重要，交货量限制。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>绿色食用色素</td><td>麦考密克</td><td> N / A </td><td>必须过滤</td></tr><tr><td>汉密尔顿玻璃注射器（100微升） </td><td>西格玛Aldrick </td><td> 20702 </td><td></td></tr><tr><td> LuerMxF主题风格的白色尼龙</td><td>小零件， </td><td> VPLF - LC78 - 1 - 25 </td><td></td></tr><tr><td>细针距注射针头</td><td>波普尔</td><td> 7111 </td><td>尺寸:33号（SWG）× ¼"（6.35毫米） </td></tr><tr><td>凌晨视透射</td><td>呼吸</td><td> 1017920 </td><td></td></tr><tr><td> 2.25X头戴放大镜</td><td> MagEyes </td><td>型号5 </td><td>选择适合个人喜好的放大倍率</td></tr></tbody></table>

delivery of therapeutic agents through intracerebroventricular (icv) and intravenous (iv) injection in mice

尽管血脑屏障的保护作用，起着屏蔽大脑，中枢神经系统（CNS）的访问限制， 设计 1,2神经退行性疾病的潜在疗法的失败中最常见的结果。神经退行性疾病，如脊髓性肌萎缩症（SMA），下运动神经元受到影响，可以极大地受益，从引进治疗药物进入中枢神经系统。此视频的目的是为了演示两种不同的注射范式传递到新生小鼠出生后不久，治疗材料。其中一个方法是直接注射入脑侧脑室（侧脑室）材料传递到中枢神经系统通过脑脊液3,4结果。第二种方法是一个时间的静脉注射（静脉注射），可以引入不同的循环系统的疗法，导致全身交付，包括中枢神经系统5。如果一个合适的病毒载体和病毒血清型是利用广泛的中枢神经系统传导是可以实现的的。颞静脉注射的可视化和利用是可行的出生后第6天。但是，如果传递的材料是为了达到中枢神经系统，这些注射应采取血脑屏障，同时由于其不成熟的状态，更渗透，最好到产后第2天之前。充分发展的血脑屏障，极大地限制了静脉交付的成效。一次手术aptitude是实现两个输送系统是简单而有效的的。他们不需要任何广泛的手术设备，可以由一个人完成。然而，这些技术并非没有挑战。产后第2天的幼崽和随后的小目标领域的体积小，可以使注射难以执行，最初具有挑战性的复制。

Watch this Scientific Journal Video about Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice at JoVE.com

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

本文演示了两种非常不同的方法:注射入脑（脑室）和2）全身（静脉注射）1）引入到中央的新生小鼠的中枢神经系统治疗药物。

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

delivery-therapeutic-agents-through-intracerebroventricular-icv

Nanyang Technological University

Research

JoVE Journal

Medicine

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

59.2K 次观看.  University of Missouri. 本文演示了两种非常不同的方法:注射入脑（脑室）和2）全身（静脉注射）1）引入到中央的新生小鼠的中枢神经系统治疗药物。

视频: Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

原位Xenografting人类荧光素酶标记的恶性外周神经鞘瘤细胞在体内测试候选治疗药物

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

科研

医学

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

在小鼠的脑出血模型:注射自体血液或细菌胶原酶

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜:对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

药理试验药物的疗效在心包目标交付模型在猪

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE基因沉默:一个七聚物型小导RNA库的筛选潜在的癌症治疗药

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

一次性充填机对治疗药物的肺吹入小动物

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

经皮超声心动图引导心肌注射液与大鼠前体细胞的临床研究

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...