Name: intravenous injections in neonatal mice
Uploaded: 2014-11-11T13:00:00.000+00:00
Duration: 5 min 17 s
Description: Watch this Scientific Journal Video about Intravenous Injections in Neonatal Mice at JoVE.com

作者要感谢SMA的NINDS，FightSMA和家庭提供财政支持。 SEGL由NINDS培训资助＃5T32NS077984-02支持。

在协议中列出的所有程序已经批准学院为美​​国俄亥俄州立大学动物使用及护理委员会（IACUC）。 1.准备工作区<ol><li>收集湿冰麻醉的小鼠幼仔，​​一个空的保持架，以分隔从垫料，在解剖显微镜，其可被定位成一定角度喷射（使用光源以90°的光源坝&#160;角向注射部位掩盖了静脉），清洁表面放置动物的注射，棉签，3/10毫升的胰岛素注射器用3/8"为30G针（每动物1）和1％伊文思蓝染料（与用于训练的磷酸盐缓冲盐水（PBS）中）的溶液制成。 </li><li>删除大坝到一个单独的笼子，而操控幼崽。 </li></ol> 2.注射过程<ol><li>直接将单小狗在湿冰30-60秒，以麻醉的动物。不要离开吨他的动物冰上过长因体温过低相关并发症，包括心室颤动，组织缺氧和代谢性酸中毒的风险。 注:我们的经验是30-60秒，足以减缓小狗的动作，让注射。如果更深麻醉需要，吸入剂，如1-2％异氟醚可以是合适的。 </li><li>而动物是冰，装入注射器30微升的伊文思蓝染料。 </li><li>当动物被完全麻醉，通过在仍呼吸冰缺乏运动的确认后，将它在显微镜下。为右旋注入，面对动物的枪口右侧。将左手的食指上枪口和左手的中指尾椎到耳芽，使耳芽是食指和中指（ 图1）之间。 </li><li>检查只是前耳芽的浅表毛细血管的运动时，皮肤被操纵。该毛细管是不是目标，但是识别是颞静脉识别非常重要。接下来，找到一个黑暗，朦胧的静脉不如保持不变，无论皮肤位置的毛细血管。颞静脉出现神出鬼没，运行背侧到腹侧，并馈入颈静脉（ 图1）。 </li><li>输入与针头斜面向上颞静脉。如果正确地插入时，也能够查看针头斜面充满血液通过皮肤。然后压下柱塞慢慢地和音符静脉向下的面一侧的热烫。 </li><li>允许针留在静脉内用于增加10-15秒，以防止注入剂的回流。 </li></ol> 3.后喷射<ol><li>正确的注射后，小狗应立即变成蓝色。取下针头和使用轻柔的力向棉棒适用于注射部位直到血块。 </li><li>监控小狗遇险的迹象。让小狗2-3分钟，以恢复和复温河ecognize当小狗是有意识的，正直和移动，久违的笼子前。世界杯研究者的戴手套的手幼仔提供适当的保暖，如果有必要，以帮助恢复。或者，将笼下一个加热垫，以方便升温注入小狗。一般的伊文思蓝染料注射后，安乐死幼仔。注射试验材料时，不要包括染料。 </li><li>将小狗放回笼子，确保小狗涂有被褥和/或nestlet确保reacceptance由大坝。 </li><li>使用新的注射器和棉棒每个小狗保持无菌。 </li></ol>

<ol>
	<li>Foust, K. D., et al. <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> Nature. 27, 59-65 (2009).</li><li>Dominguez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+scAAV9+delivery+of+a+codon-optimized+SMN1+sequence+rescues+SMA+mice.">Intravenous scAAV9 delivery of a codon-optimized SMN1 sequence rescues SMA mice.</a> Human molecular genetics. 20, 681-693 (2011).</li><li>Rahim, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+AAV2/9+to+the+fetal+and+neonatal+mouse+leads+to+differential+targeting+of+CNS+cell+types+and+extensive+transduction+of+the+nervous+system.">Intravenous administration of AAV2/9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system.</a> FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 25, 3505-3518 (2011).</li><li>Foust, K. D., et al. <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>Duque, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons.">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol Ther. 17, 1187-1196 (2009).</li><li>Bostick, B., Ghosh, A., Yue, Y., Long, C., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+AAV-9+transduction+in+mice+is+influenced+by+animal+age+but+not+by+the+route+of+administration.">Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration.</a> Gene Ther. 14, 1605-1609 (2007).</li><li>Miyake, N., Miyake, K., Yamamoto, M., Hirai, Y., Shimada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+transfer+into+the+CNS+across+the+BBB+after+neonatal+systemic+delivery+of+single-stranded+AAV+vectors.">Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors.</a> Brain research. 1389, 19-26 (2011).</li><li>Porensky, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+administration+of+morpholino+antisense+oligomer+rescues+spinal+muscular+atrophy+in+mouse.">A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse.</a> Human molecular genetics. 21, 1625-1638 (2012).</li><li>Hua, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+SMN+restoration+is+essential+for+long-term+rescue+of+a+severe+spinal+muscular+atrophy+mouse+model.">Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.</a> Nature. 478, 123-126 (2011).</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> Journal of the American Association for Laboratory Animal Science : JAALAS. 46, 50-54 (2007).</li><li>Glascock, J. J., et al. <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+therapeutic+agents+through+intracerebroventricular+(ICV)+and+intravenous+(IV)+injection+in+mice.">Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice.</a> J. vis. Exp. (56), (2011).</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> Laboratory animal science. 49, 328-330 (1999).</li><li>Bevan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+heart+failure+in+the+SMNDelta7+model+of+spinal+muscular+atrophy+and+correction+by+postnatal+scAAV9-SMN+delivery.">Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery.</a> Human molecular genetics. 19, 3895-3905 (2010).</li><li>Valori, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+delivery+of+scAAV9+expressing+SMN+prolongs+survival+in+a+model+of+spinal+muscular+atrophy.">Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy.</a> Science translational medicine. 2, (2010).</li><li>Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+intraperitoneal+or+intravenous+injections+of+recombinant+adeno-associated+virus+type+8+transduce+dorsal+root+ganglia+and+lower+motor+neurons.">Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.</a> Hum Gene Ther. 19, 61-70 (2008).</li><li>Guggenbuhl, P., Grosbois, B., Chales, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaucher+disease.">Gaucher disease.</a> Joint, bone, spine : revue du rhumatisme. 75, 116-124 (2008).</li><li>Daly, T. M., Vogler, C., Levy, B., Haskins, M. E., Sands, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+gene+transfer+leads+to+widespread+correction+of+pathology+in+a+murine+model+of+lysosomal+storage+disease.">Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease.</a> Proc Natl Acad Sci U S A. 96, 2296-2300 (1999).</li><li>Daly, T. M., Ohlemiller, K. K., Roberts, M. S., Vogler, C. A., Sands, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+systemic+clinical+disease+in+MPS+VII+mice+following+AAV-mediated+neonatal+gene+transfer.">Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer.</a> Gene Ther. 8, 1291-1298 (2001).</li><li>Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juvenile+neuronal+ceroid+lipofuscinoses.">Juvenile neuronal ceroid lipofuscinoses.</a> Advances in experimental medicine and biology. 724, 138-142 (2012).</li><li>Forsythe, E., Beales, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bardet-Biedl+syndrome.">Bardet-Biedl syndrome.</a> European journal of human genetics. 21, 8-13 (2013).</li><li>Bevan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+gene+delivery+in+large+species+for+targeting+spinal+cord,+brain,+and+peripheral+tissues+for+pediatric+disorders.">Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders.</a> Mol Ther. 19, 1971-1980 (2011).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab animal. 40, 155-160 (2011).</li><li>Saunders, N. R., Joakim Ek, C., Dziegielewska, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neonatal+blood-brain+barrier+is+functionally+effective,+and+immaturity+does+not+explain+differential+targeting+of+AAV9.">The neonatal blood-brain barrier is functionally effective, and immaturity does not explain differential targeting of AAV9.</a> Nature biotechnology. 27, 804-805 (2009).</li><li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+AAV9-mediated+suppression+of+mutant+SOD1+slows+disease+progression+and+extends+survival+in+models+of+inherited+ALS.">Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.</a> Mol Ther. 21, 2148-2159 (2013).</li></ol>

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血管内递送药物到中枢神经系统或整个身体难以在疾病的新生鼠模型。所描述的协议，是静脉内给药溶液加入到新生小鼠以最小的设备要求一种快速，相对非侵入性的方式。虽然颞面部静脉可以通过肉眼观察，注射液可以具有更高的准确性与使用显微镜和光纤光源的，特别是对于一个不熟练的喷射器。在新生小鼠的血管内注射剂具有较高的成功率12，和AAV转导已经在早幼崽被观察到即使注入剂的一部分被皮下给药。注射用鼠标幼仔的耐受性良好，并在FVB / N或C57BL / 6背景水坝容忍操纵的幼崽非常好。但是，在极少数情况下，水坝可以摧毁整个窝。这些注射剂可以在小鼠中一样小0.8克通过1.6克成功进行。另一种注射液n对于新生小鼠的路线已经公布了美国和其他国家，包括眼眶，intrajugular和腹腔10,15,22。我们优选在这里所描述的注射途径，因为它是临床上相关的，能够通过单一的实验者没有特殊装备的要求进行，并允许一个宽范围体积的待施用。 一个常见的​​问题有经验的时候进行注射是无法查看的时间静脉，因此错位针。光纤光源应放置在&lt;90°，正在举行的静脉最佳观看时的头向下闪耀。喷油器应该花时间来调整光源，以满足他们的需求。颞静脉观测显著下降如果幼仔过大，表征为较旧然后P2或大于2.0克第二个问题可能是确定针头斜面进入静脉。颞面部静脉是superfiCIAL，所以必须小心，以避免进入静脉的下方。确保针头斜面进入平行于静脉将防止通过静脉穿刺。针的位置是最好的缓慢注射和静脉后续漂白验证。 颞浅静脉是在头部的两侧可见。左撇子人可能会发现，面对动物研究者的左边可能是符合人体工程学的更好，应该为每个单独的调查来确定。应静脉不可见或不可被错误定位于初始的尝试中，针对静脉在头部的相对侧上是合适的。如果静脉被刺破的头部的两侧上，可注射到第二位置期间通过所述第一现场发生一些泄漏。根据显微镜的工作距离，也可适当构筑小平台，在其向动物注射;例如，一个空的泡沫塑料在50毫升的锥形容器管包装（ 图1）。这使得研究者以定位平台的边缘附近的动物，使得研究者的注入手可以放在桌子上直接休息。这样可以提高进入静脉的角度和最小化光阻塞。 有一些限制的协议，包括注射量，患者的年龄窗口用于注射和坝响应的不可预测性的效用。进样体积为幼仔被限制为50微升。之前我们已经给予动物100微升，但已观察到增加注射相关的死亡率相比，容量注射剂低，但其他人报告说成功交付100微升10,12。此外，已经表明，增加的注射体积可以破坏血脑屏障23。常的实验试剂用PBS混合，以保持恒定的50微升注射体积。我们不知道研究examini纳克注射体积对扩散的效果在整个新生儿小鼠和经验研究应该被执行以确定在给定的剂​​量下对注射量为新的试剂的效果。第二个限制是，颞浅静脉是在第一和第二日龄容易看见。改变动物的大小，皮肤色素沉着和厚度相结合，以达到掩盖静脉后世虽然等报告，通过6 10,12日龄进入静脉。另外，我们用超声引导下注射强心提供病毒载体的流通P5和P10动物4。最后，该过程的鼠标水坝的接受是不可预测的。我们已经处理FVB / N，C57BL / 6和B6SJL小鼠跨越神经肌肉疾病和孤独症谱系障碍的小鼠模型。 FVB / N小鼠是最宽容的，而菌株对C57BL / 6背景偶尔会破坏个人的幼崽或整个窝，但这种情况极少发生。然而，这规划足够的n表示实验时，应该是一个考虑因素。大坝验收通过什么病的小鼠品系的造型得到进一步的影响。例如，自闭症谱系障碍的模型具有高得多的速率小狗排斥的基础上，本底应变超过预期。培育幼崽可能是一个考虑因素，如果大坝接受是一个问题。其他方法来限制​​排斥包括执行从大坝一个单独的房间打针来限制访问的小狗超声波发声，并擦大坝的鼻子和/或用布蘸幼崽用70％的乙醇，以掩盖任何调查的气味。幼崽也应擦脏寝具接受帮助。 此协议已被成功地用于治疗SMA的小鼠模型。形状记忆合金是一种小儿神经变性疾病，主要影响脊髓的下运动神经元。如果不进行治疗，这种小鼠模型诞生平均死亡后1​​5天。 AFAAV基因治疗的1日龄小鼠器静脉注射，进展生存期延长至一年，证明所描述的注射方案2,4,14的安全性和耐受性。在相同的SMA的小鼠模型以下IV注射反义寡核苷酸8,9类似的结果也得到了。未来的应用包括递送的其他细胞和基因疗法，以及建立新生儿模型对中枢神经系统和外周的发育障碍给外源性表达的cDNA或RNA干扰盒24。静脉注射交付的批准的临床方法;因此，利用这种技术可以在对各种试剂的临床前研究中是有利的。

递送治疗剂到在儿科疾病的鼠模型中的中枢神经系统（CNS）中的仍然是一个挑战。小鼠模型中的新生疾病状态是矮小和发育不成熟，因此可能难以直接注入在中枢神经系统内的适当的结构。血管内注射的治疗剂是一种非侵入性的，良好耐受的方法，提供的细胞，药物，或病毒载体的整个身体，包括中枢神经系统1-5和视网膜3,5-9。以前的出版物描述了使用透10,11颞面部静脉注射，无需解剖镜下11,12，还是需要两个人来注入10。在这个协议中所描述的注射技术是有利的，因为一个人可以注入幼仔，和光源，以查看颞静脉没有触摸小狗，省去了手术带或小狗的连接到一个固定的表面诸如透射11。输送的腺相关病毒载体的血清型9（AAV9）在小鼠中产生强大的表达在神经元和星形胶质细胞的整个大脑和脊髓（ 图1）。血管内递送病毒载体到颞浅面静脉已被可靠地使用在各种研究中新生小鼠治疗小儿神经肌肉病脊髓性肌萎缩（SMA），2,4,13,14，并最终增加了处理的小鼠的寿命。 血管内注射新生小鼠也有效地靶向外周神经系统和外周器官（ 图2）。以下注射的AAV，背根神经节，肝脏，心脏，骨骼肌，肺和肠的肠肌丛的转导已观察1,3,6,7,15。中枢神经系统和周围的广泛传导，使理想的注射这种方法需要对全球的疾病表现转基因，比如高雪氏病16和其他溶酶体贮积病17,18，巴滕病和相关的神经元蜡样lipofuscinoses，19和巴比二氏综合征，一种遗传性多系统疾病发病的早期儿童20出现的症状。血管内注射到新生小鼠中，也应考虑作为新的建模系统范围的儿科疾病的方法。这种技术已被翻译为较大的动物模型5,21和血管内注射已经存在作为递送治疗剂的临床上可接受的方法。 目前的协议描述了通过颞浅静脉脸上交付代理商新生小鼠不晚于产后第2天注射一种简单，有效的方法，可以通过一个单一的完成，实行个人和既受两个幼崽和水坝的耐受性。幼崽体验最小痛苦和快速恢复。 ImportanTLY，成功的注入将导致管理代理的全球交付。这个协议是适当的递送病毒载体，药物制剂或细胞对新生小鼠。

<table><tbody><tr><td>Thinpro insulin syringe</td><td>Terumo</td><td>SS30M3009</td><td>3/10 cc, 3/8&quot; needle, 30 G, 1 per mouse</td></tr><tr><td>Evans blue dye</td><td>Sigma-Aldrich</td><td>E2129</td><td>Dilute to 1% with 1x Phosphate Buffered Saline</td></tr><tr><td>Cotton tipped applicators</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Fiber optic light source</td><td>Fisher Scientific</td><td>12-562-36</td><td></td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td></td></tr></tbody></table>

intravenous injections in neonatal mice

静脉注射是一种临床应用的方式提供治疗。对于成年啮齿类动物和大型动物，静脉注射在技术上是可行的程序。然而，一些小鼠模型可以有早发性疾病具有病情进展快，使潜在疗法的管理难度。的时间（或面部）静脉只是前耳芽在小鼠和显然对于前两天可见在出生后的头使用解剖显微镜的任一侧。在此窗口中，颞静脉可以与体积可达50微升注射。注射是安全的，由两个幼仔和堤坝的耐受性良好。一种典型的注射过程中1-2分钟，在此之后，小狗返回到笼中完成。到了第三日龄静脉是很难想象和注射过程在技术上变得不可靠。这一技术已被用于递送的腺相关病毒（AAV）VECT口服补液盐，这反过来又可以用于根据所述病毒血清型所选择的动物的生活提供几乎全身性的，稳定的转基因表达。

在适当的注射，静脉应暂时关闭清楚，或者焯烫。如果注射染料整个小狗应该在几秒钟内变成蓝色。如果发生了不正确的喷射，往往有一个浓缩皮下推注的头部或颈部和注入剂可能泄漏的注射部位的出。不当注射剂也可能会导致围绕喉部青紫的外观。接收皮下注射（ 即注射液没有充分的静脉交付），一般遇到任何不良副作用和常规响应处理的正常小鼠注射做幼仔。 根据递送的治疗，成功的注射可导致广泛递送至中枢神经系统和外周器官。当注入自免费腺相关病毒血清型9表达绿色荧光蛋白（GFP）与鸡β肌动蛋白的杂合启动子（CB）（一起称为"的scAAV9 CB GFP"），反基因表达是在跨越多个脑区域的神经胶质细胞和神经元和脊髓（ 图2A-2C）中找到。血管内注射的scAAV9 CB的GFP还导致广泛表达在整个周边上，有明显的表达在心脏和肝脏组织（ 图3A-3D）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52037/52037fig1highres.jpg" width="500" /> 图1:（a）在临时搭建的平台，注入从吸湿垫（"尿布"）取得了50毫升锥形包装缠泡沫塑料架。该平台允许研究者休息他们的手放在邻近于平台的表。这使得调查人员在平坦的角度相对于鼠标的小狗一个执行注射。（B）图像显示的食指和中指之间的小狗固定。（ <stron克&gt; C）图像显示cryoanesthesia后，P2的小狗。静脉注射是由白色箭头指示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52037/52037fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52037/52037fig2highres.jpg" width="500" /> 图2:中枢神经系统的基因表达以下新生儿静脉注射自互补AAV9表达绿色荧光蛋白（CB的scAAV9 GFP）产后第1天（P1）的乳鼠静脉注射自1×10 11载体基因组（VG）。互补（SC）的scAAV9 CB GFP。四星期后喷射，处死小鼠并灌注4％多聚甲醛。组织标本，切片和染色的GFP表达。 GFP的表达被认为是整个大脑，包括前纹状体（A）通过量小时脑桥小脑和（B）。 GFP阳性细胞通常是神经元和星形胶质细胞。 GFP的表达是在脊髓（C）的同时检测。脊髓运动神经元和背根神经节神经元用新生儿IV交付有效地转。 GFP阳性血管和星形胶质细胞也被视为整个脊髓灰质。所有比例尺= 200微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52037/52037fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52037/52037fig3highres.jpg" width="500" /> 图3:GFP表达在以下新生儿静脉注射自我互补AAV9的GFP（的scAAV9 CB GFP）的 GFP的免疫荧光是可检测的，在成年小鼠心脏（A）和肝（C）的外周器官。 </str翁&gt;以下新生儿静脉注射的scAAV9 CB GFP的。为了进行比较，GFP的表达是不存在于未注射的小鼠的心脏（B）和肝脏（D）中 。所有比例尺= 100微米。

Watch this Scientific Journal Video about Intravenous Injections in Neonatal Mice at JoVE.com

Intravenous Injections in Neonatal Mice

儿科疾病的动物模型可以体验早期发病和严重的疾病进展。临床相关治疗交付给年轻小鼠模型可以在技术上难度。这个协议描述了一种非侵入性的静脉内注射的方法在生命的最初2日龄新生小鼠。

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Universidad San Sebastian

Research

JoVE Journal

Biology

58.2K 次观看.  Ohio State University. 儿科疾病的动物模型可以体验早期发病和严重的疾病进展。临床相关治疗交付给年轻小鼠模型可以在技术上难度。这个协议描述了一种非侵入性的静脉内注射的方法在生命的最初2日龄新生小鼠。 

视频: Intravenous Injections in Neonatal Mice

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

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 often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

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

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

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 ...

科研

医学

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

使用<em&gt;在体内</em&gt;影像监测进展的实验小鼠巨细胞病毒感染的新生儿

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

免疫与感染

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

小鼠宫内移植中静脉和羊膜内植入的研究

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

发育生物学

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

神经科学

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

使用徒手方法对小鼠进行脑室内注射

Free-Hand Intracerebroventricular Injections in Mice

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. An intracerebroventricular (ICV) injection allows the widespread delivery of the experimental agent throughout the brain (particularly in the structures near the ventricles). Here, methods for making free-hand ICV injections in adult mice are described. By using visual and tactile landmarks on the heads of mice, injections into the lateral ventricles can be made rapidly and reliably. The injections are made with a glass syringe held in the experimenter&#39;s hand and placed at approximate distances from the landmarks. Thus, this technique does not require a stereotaxic frame. Furthermore, this technique requires only brief isoflurane anesthesia, which permits the subsequent assessment of mouse behavior and/or physiology in awake, freely behaving mice. Free-hand ICV injection is a powerful tool for the efficient delivery of experimental agents into the brains of living mice and can be combined with other techniques such as frequent blood sampling, neural circuit manipulation, or in vivo recording to investigate neuroendocrine processes.

作者聚焦：生殖神经控制的见解、创新和未来途径 

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is described.

小鼠徒手脑室内注射

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

A Neonatal Rodent Model of Retroorbital Vein Injection

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an important method for compound administration in research, where successful proof-of-concept studies can progress into much-needed neonatal clinical trials. Most intravenous studies in neonatal rodents use the superficial temporal/facial vein. However, retroorbital injection becomes unreliable in neonatal rodents older than 2 days after the skin darkens and the vein is no longer visible. In the present protocol, we describe the retroorbital injection of the venous sinus in both the neonatal mouse and rat at ages when the superficial temporal vein is no longer visible, but the eyes have not opened yet. Eye-opening facilitates retro-orbital injection by enabling the researcher to clearly see that they are not perforating the eye when inserting the needle. We demonstrate that this technique can be performed in a reliable and reproducible manner without adverse effects. Additionally, we show that it can be used in many studies, such as administering compounds to study neonatal brain injury.

This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

眶后静脉注射的新生儿啮齿动物模型

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an ...

在新生小鼠中进行鞘内注射的综合方案

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

作者聚焦: 鞘内注射 – 一种高效可靠的递送方法，用于测试新生小鼠大脑中基因编辑的功效

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

新生小鼠鞘内注射用于基因组编辑和药物递送

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

在新生小鼠静脉注射

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交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

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小鼠宫内移植中静脉和羊膜内植入的研究

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病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

小鼠徒手脑室内注射

小鼠徒手脑室内注射

小鼠徒手脑室内注射

眶后静脉注射的新生儿啮齿动物模型

新生小鼠鞘内注射用于基因组编辑和药物递送