著者は、財政支援のためのNINDS、FightSMA、とSMAの家族を認識したい。セグルはNINDS訓練助成金＃5T32NS077984-02によってサポートされています。

プロトコールに記載されているすべての手順は、オハイオ州立大学の動物使用とケア委員会（IACUC）研究所承認されている。 ワークスペースの調製<ol><li>ごみ、解剖顕微鏡、90℃で注入（光源の使用に対してある角度で配置することができ、光源からのダムを分離するために、空のケージの仔マウスを麻酔湿式氷を集める&#160;注射部位に角度静脈を不明瞭）、注射のために動物を配置する清浄な表面、綿棒、3/8 &quot;30 G針（動物あたり1つ）および1％エバンスブルー色素（と3/10 ccのインスリン注射器訓練のためのリン酸緩衝生理食塩水（PBS））溶液を用いて作った。 </li><li>子犬を操作しながら、別々のケージにダムを削除します。 </li></ol> 2.注入手順<ol><li>動物を麻酔30-60秒間濡れた氷の上に直接、単一の子犬を置きます。トン放置しないでください彼による心室細動、組織低酸素および代謝性アシドーシスなどの低体温症に関連する合併症の危険性への氷の上で動物が長すぎる。 注：私たちの経験は、30〜60秒の注入を可能にするために、子犬の動きを遅くするのに十分であることである。深い麻酔が必要な場合、そのような1〜2％イソフルランなどの吸入剤が適切であり得る。 </li><li>動物が氷の上ですが、エバンスブルー染料30μlのシリンジをロードします。 </li><li>動物が完全に麻酔されたとき、まだ呼吸しながら、氷の上での動きの欠如によって確認された顕微鏡下に移動します。右利きの注射のために、右に動物の銃口に直面しています。耳の芽は人差し指と中指（ 図1）の間になるよう、耳の芽に銃口を左人差し指と左中指の尾を置きます。 </li><li>皮膚が操作されたときに移動する表面的な毛細血管のための耳の芽にちょうど前方を調べます。このキャピラリーはしかし、対象ではありません識別は、時間的な静脈認証のために重要である。次に、関係なく、肌の位置の固定されたままでキャピラリーに劣って暗い、闇の静脈を探します。時間的な静脈が影の表示され、腹側に背を実行し、頸静脈（ 図1）に供給されます。 </li><li>針のベベルを上にして一時的な静脈を入力してください。正しく挿入された場合は、針のベベルを皮膚を介して血液を充填見ることができる。その後、ゆっくりとプランジャーを押し下げるとノートでは、顔の側を下静脈のブランチング。 </li><li>針が注入物質の逆流を防ぐために追加された10〜15秒間の静脈内にとどまることを可能にする。 </li></ol> 3.ポストインジェクション<ol><li>適切な注入後、子犬は、ほとんどすぐに青色に変わるはずです。針を外して、血の塊になるまで注射部位に綿棒を適用するために穏やかな力を使用しています。 </li><li>苦痛の徴候のために子犬を監視します。子犬2-3 minは回復しrewarmすることを許可する、R子犬がケージに戻る前に、意識的な直立と移動しているときecognize。カップは研究者の手袋をはめた手の中に子犬は、必要に応じて、回復を支援するために、適切な暖かさを提供します。代わりに、注入された子犬を温める容易にするために、ホームケージの下に加熱パッドを配置。一般的にエバンスブルー色素注入後、子犬を安楽死させる。試験材料を注入する際に染料を含めないでください。 </li><li>バックホームケージに子犬を置き、子犬がダムによってreacceptanceを確保するために、寝具、および/またはnestletでコーティングされていることを確認してください。 </li><li>無菌性を維持するために、各子犬のための新しい注射器と綿棒を使用してください。 </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. 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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. 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著者らは、開示することは何もない。

CNSまたは全身の薬剤の血管内送達は、疾患の新生児マウスモデルでは困難である。記載されているプロトコルは、静脈内に、最小限の設備要件を新生児マウスにソリューションを管理するための迅速、比較的非侵襲的な方法です。時間的な顔の静脈が肉眼で見ることができるが、注射は、特に経験の乏しいインジェクタ、顕微鏡および光ファイバ光源を用いて、より高い精度を有することが...

小児疾患のマウスモデルにおいて、中枢神経系（CNS）への治療薬の送達は課題である。モデル新生児疾患状態が過小と発生的に未熟であるため、直接CNS内の適切な構造体に注入することが困難であることをマウス。治療薬の血管内注入がCNS 1-5および3,5-9網膜を含む全身への細胞、薬物、またはウイルスベクターを送達するための非侵襲性、忍容性が良好な方法である。以前の刊行物は、解剖顕微鏡11,12なしで、イルミ10,11を使用して、一時的な顔の静脈注射、または記述10を注入する二人の個人を必要とする。一個人が子犬を注入することができ、光源が時間的静脈子犬に触れていない表示する、サージカルテープの必要性や、固定表面への子犬の付着を排除するため、このプロトコルに記載注入技術が有利であるイルミ11など。マウスでのアデノ随伴ウイルスベクター血清型9（AAV9）の送達は、脳や脊髄（ 図1）を通じて、ニューロンおよびアストロサイトでの強い発現を生成します。浅側頭動脈顔面静脈へのウイルスベクターの血管内送達は、確実に、小児神経筋疾患脊髄性筋萎縮症（SMA）2,4,13,14を治療するための新生仔マウスの様々な研究で使用され、最終的に処理されたマウスの寿命を増加している。 新生児マウスの血管内注入も効果的に末梢神経系および末梢器官（ 図2）標的とする。 AAVの注入後、後根神経節、肝臓、心臓、骨格筋、肺、および腸の筋層間神経叢の形質導入は、1,3,6,7,15観察されている。 CNSおよび末梢の広範な伝達は、グローバルな表現を必要とする疾患の注入のこの方法は最適ですそのようなゴーシェ病16および他のリソソーム蓄積症17,18、バッテン病と関連する神経細胞セロイドlipofuscinoses、19およびバルデ·ビードル症候群、幼児20で発生する症状の発症との遺伝的多臓器障害などのトランスジーン、。新生仔マウスへの血管内注入はまた、モデリングシステム全体の小児疾患の新規な方法と考えるべきである。この手法は、より大きな動物モデル5,21に翻訳され、血管内注射は、すでに治療薬を送達することが臨床的に許容される方法として存在する。 現在のプロトコルは、生後2日目注射より遅くとも浅側頭顔静脈から新生児マウスに薬剤を送達しない、シンプルで効率的な方法を記述する単一完成させることができる、個々の練習、よく子犬やダムの両方によって許容されている。子犬は、最小限の苦痛を経験し、迅速に回復。 ImportanTLY、成功した注射が投与された薬剤のグローバルなデリバリーになります。このプロトコルは、ウイルスベクター、新生マウスに薬剤または細胞の送達に適している。

<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

静脈内注射は、治療薬を提供するために臨床応用可能な方法である。大人の齧歯類および大型動物の場合は、静脈内注射は技術的に可能と日常的なものである。しかし、いくつかのマウスモデルは、潜在的な治療薬の投与が困難に急速進行性疾患の早期発症を持つことができる。時間的（または顔面）静脈がちょうどマウスにおいて、耳の芽の前方および解剖顕微鏡を用いて、頭部の両側に、出生後、明らかに最初の2日間は表示されている。このウィンドウ中に、一時的な静脈を50μlまでの容量で注射することができる。注射は安全で忍容子犬やダムの両方によって許容される。典型的な注射手順は子犬をホームケージに戻された後、1〜2分以内に完了する。第生後によって静脈視覚化が困難であり、注入手順は、技術的に信頼できないとなる。この技術は、アデノ随伴ウイルス（AAV）のvectの送達のために使用されている順番に選択されたウイルスの血清型に応じて、動物の生活のため、ほぼ身体全体で、安定的な導入遺伝子発現を提供することができます論理和をとり、。

適切な注入中に、静脈が瞬​​間的にクリア、または湯通し有効にしてください。染料注入した場合、全子犬が数秒以内に青色に変わるはずです。不適切な注入が発生した場合、頭部または頸部および注入物質中の濃縮皮下ボーラス注射部位から漏出してもよいことが多い。不適切な注射はまた、喉の周りにあざが出現する可能性があります。皮下注射を受けるの仔（ すなわち 、注?...

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

Intravenous Injections in Neonatal Mice

小児疾患の動物モデルは、早期発症と積極的な疾患の進行を体験することができます。臨床的に若いマウスモデルに関連する治療送達は技術的に困難であることができる。このプロトコルは、人生の最初の2日以内に出生後の新生児マウスのための非侵襲的静脈内注入法を説明しています。

新生児マウスにおける静脈注射

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

58.0K ビュー.  Ohio State University. 小児疾患の動物モデルは、早期発症と積極的な疾患の進行を体験することができます。臨床的に若いマウスモデルに関連する治療送達は技術的に困難であることができる。このプロトコルは、人生の最初の2日以内に出生後の新生児マウスのための非侵襲的静脈内注入法を説明しています。 

ビデオ: 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.

マウスの脳室内（ICV）と静脈（IV）注入による治療薬のデリバリー

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 ...

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.

活用<em生体内で&gt;</em新生児の実験マウスサイトメガロウイルス感染の進行を監視する&gt;イメージング

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

免疫・感染学

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.

マウス モデルにおける静脈内投与と胎嚢内子宮内移植

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 ...

発生生物学

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.

永続かつ広範な神経伝達のための新生児マウス脳の脳室内ウイルス注射

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

神経科学

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.

新生児の脳へのウイルス粒子と蛍光マイクロビーズの脳室内および血管内注入

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 ...

フリーハンドアプローチを用いたマウスにおける脳室内注射の実施

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.

著者スポットライト:生殖神経制御における洞察、イノベーション、および将来の道筋 

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.

マウスにおけるフリーハンド脳室内注射

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.

眼窩後静脈注射の新生児げっ歯類モデル

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

新生児マウスに髄腔内注射を実施するための包括的なプロトコル

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.

著者スポットライト:髄腔内注射 – 新生児マウスの脳における遺伝子編集の有効性をテストするための効率的で信頼性の高い送達方法

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

ゲノム編集とドラッグデリバリーのための新生児マウスの髄腔内注射

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

新生児マウスにおける静脈注射

要約

さらに動画を探す

この動画の章

マウスの脳室内（ICV）と静脈（IV）注入による治療薬のデリバリー

活用

マウスモデルにおける静脈内投与と胎嚢内子宮内移植

永続かつ広範な神経伝達のための新生児マウス脳の脳室内ウイルス注射

新生児の脳へのウイルス粒子と蛍光マイクロビーズの脳室内および血管内注入

マウスにおけるフリーハンド脳室内注射

マウスにおけるフリーハンド脳室内注射

マウスにおけるフリーハンド脳室内注射

眼窩後静脈注射の新生児げっ歯類モデル

ゲノム編集とドラッグデリバリーのための新生児マウスの髄腔内注射

新生児マウスにおける静脈注射

要約

さらに動画を探す

この動画の章

マウスの脳室内（ICV）と静脈（IV）注入による治療薬のデリバリー

活用

マウス モデルにおける静脈内投与と胎嚢内子宮内移植

永続かつ広範な神経伝達のための新生児マウス脳の脳室内ウイルス注射

新生児の脳へのウイルス粒子と蛍光マイクロビーズの脳室内および血管内注入

マウスにおけるフリーハンド脳室内注射

マウスにおけるフリーハンド脳室内注射

マウスにおけるフリーハンド脳室内注射

眼窩後静脈注射の新生児げっ歯類モデル

ゲノム編集とドラッグデリバリーのための新生児マウスの髄腔内注射

マウスモデルにおける静脈内投与と胎嚢内子宮内移植