Name: in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow
Uploaded: 2015-02-04T15:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

The authors would like to thank Dr. George Gittes for advice and expertise throughout this study. SSL was supported by an American Heart Association fellowship (11POST7330002). Further to this SSL and this study was supported by an NIDDK Mentored Research Scientist Development Award (DK096996) and by the Children&#8217;s Hospital of Pittsburgh.

注：ピッツバーグ大学の施設内動物管理使用委員会は、すべての実験を承認した。 超音波マイクロインジェクションインスツルメンツおよび胚の作製<ol><li>ステージを設定し、マウントして、プローブ（ 図1）と同様に手術器具（ 図2）。 37℃の加温槽内の場所のリン酸緩衝生理食塩水（PBS）溶液（pH7.4）で。そのベースを介して、第二の柔軟な25 G針に付着した1mlシリンジを使用して、鉱油を完全にマイクロインジェクション針を埋める。 </li><li>回転上に針を修正アームをマウントし、鉱油溶液の空の針。 TL溶液を2.5μlの再埋める。気泡が注射針内に存在しないことを確認してください。離れてステージから、壁に向かって針アームを回転させます。 </li><li>イソフルランの連続注入を介して麻酔室で妊娠中の母親を麻酔。母親が無意識にレンダリングされると、C上に位置する鼻チューブに麻酔を転送audalステージの側面、そして完全に麻酔をかけた状態の継続を可能にするために鼻のチューブに鼻と仰臥位で母親を置く。 </li><li> 45°の角度で、または手や足にテープ手足は休息と頭上ECG /温度モニタのタブを配置。これは、妊娠中のダムが継続的にその麻酔を確実に監視することが重要であるのに十分であり、眼への印加中にその軟膏は、乾燥を軽減する。 </li><li>優しく余分な製品と毛を除去するために70％エタノール飽和綿棒で再度乾燥した綿棒で拭き取っ、そして、母親の下腹部全体に脱毛製品を適用します。切開部位（ 図3）で、すべての髪のオフ腹部の皮膚をきれいにすることを確認してください。 </li><li>細かい外科鉗子やハサミを使って妊娠中の母親に開腹を行います。すべての主要な血管や内臓器官を切断しないように注意してください。 1.5〜2センチメートル膣の上に、第一表皮のストレート切開を作成し、approximatためのリブに向けてカットを継続エリー2-3センチ。内臓器官および子宮球形嚢の内部を露出させるために、同じ長さに沿って、皮下膜を公開白線（「白線」）（ 図3）を探して、最初の切開に平行に切断。 </li></ol>胚の2.抽出<ol><li>母と胚を操縦するために6インチの綿棒アプリケーターを使用してください。静かに切開口から最初の胚を操作するアプリケータを用いて皮膚に（強制しない）押してください。最初の胚球形嚢の抽出後、静かにゆっくりと通過して、母親の外側に子宮角の残りの部分を引っ張る。この段階で切開を通って腸または他の臓器を引っ張っては避けてください。 </li><li>胚を定量化し、優しく母親に戻す左子宮角に配置します。そして、ホーンに膣から胚球形嚢遠いから始まって下方向に移動バック母に右子宮角を配置し始める。唯一の2胚を露出されたままになるまでこれを続けます（FIグレ4）。最後に、切開線に上記と平行の列の位置の胚は、子宮の循環を遮断しないように認識している。 </li><li>粘土ブロックと腕と体の間の位置に配置し、だけでなく、足や尾。粘土表面は開腹切開とほぼレベルであることを確認してください。 37℃のPBSで有窓ペトリ皿を湿ら。 </li><li>左手（胚に開窓パラレル）で右手と有窓シャーレに鉗子を拡張つかみ、ペトリ皿の上から、窓を通して〜5cmのクローズ鉗子を持って来る。完全に開窓メッシュを引き裂くことなくオープン鉗子。 </li><li>胚の上に手を操縦し、2胚球形嚢に視力を集中。なし（または軽く）スリットを介してそれらをスライドさせ、母親の腹部上にシャーレを設定し、開窓メッシングまたは鉗子で球形嚢に触れる。鉗子はまだ延長すると、いずれかの側に、それぞれの胚のベース（ 図5でそれを着座させる有窓メッシュを操作する</st栄&gt;）と、スリット開口部から鉗子を引き出します。 </li><li>次に、（ 図6）つまんまたは胚を傷つけることなく、シャーレに壁を含む青いゴムを配置します。確認粘土ブロックはしっかりとペトリ皿、胚、およびゴム含有壁をバランスされていることを確認。有窓メッシングで漏れを制御しながら、胚が完全に、（ 図6）水没するまで最後に、37℃のPBSでペトリ皿を埋める。 </li></ol> 3.注入手順<ol><li>次に、下部注入母胚ダウンZレベル調整つまみ（ 図1）を使用して、ステージとプローブ先端左と下針1/2センチで、注射針を回転させ、アームをマウントし、超音波プローブに直接並ぶ（ 図7） 。 45°離れたプローブからの針アーム（しない針）を押します。皿または超音波プローブを用いて針の先端をヒットしないように注意してください。 </li><li>超音波pで、元の高さに噴射ステージを上げる直接プローブがわずかに沈めなければならない胚上方および胚組織3-4 mm以内ローブ。使用X＆Yステージ調整ノブ（ 図8）体系的に破って、胚の心を見つけるための胚/母​​/ステージ位置を修正する。 </li><li>直接超音波画面の中央には、描かれた黒、センター標的マーカー（*）を観察する。 （ 図8）X＆Yステージ調整ノブを使用して、左心室（望ましい）またはアトリウムを見つけて、超音波画面上の黒いセンターマーカーの上に正確に注入ターゲットを配置するために、ステージ上の回転ノブを使用して、ステージを上げるか、下げる。 </li><li>超音波画面から、右に胚を移動するXステージ調整ノブを使用してください。 1/2離れてからCMと、超音波プローブに90°（ 図7）、マイクロインジェクション針の位置を変更し、元の位置に戻し腕。 </li><li>マイクロインジェクションを使用すると、アル明らかに調整つまみ（ 図9C-G）、先端を位置の針をマウントセンターマーカーでigned。針の先端が正しい平面内に集束されるように（ 図9CおよびEGでノブを使用して）噴射角を調整し、マイクロニードルのX、Y、およびZベクター。他の寸法を調整しないように注意し、単に「注射」ノブ（ 図9F）を使用して、マイクロインジェクション針を撤回。 </li><li>ここでも、正確に超音波画面上の中心マークのみにターゲットを持つバックプローブ下のX微調整段階つまみ移動胚を、使用して。左心室が全く同じX、Y、Zの平面上になっていることを確認してください。 </li><li>次に、マイクロインジェクションに単に「注射」ノブでマウント、ゆっくりとマイクロインジェクション針で胚を穿刺し、徐々に左心室に先端をもたらす。 「空」を保持し、（ 図2A＆B）いったん「埋める」をタップして、左心室にまたは空の警告ビープ音がするまで、TL·ソリューションの完全な2.5μLを注入。現時点では注射の心臓の室に入るTL（ 図10）である針の先端から発せられる影を観察する。逆に、すぐに注入が完了したときにマイクロインジェクションの「注射」ノブ（ 図9F）がマウント回転マイクロインジェクション針を撤回。 </li><li>次の胚に進みとこの一連の以下の残りの胚について、この手順を繰り返し、最終的に戻ってダムの腹部への最終胚を置きます。チャンバーボックスに麻酔を切り替えて、静かに最後の注射の時から15分間、ここで母を移動します。 </li></ol> 4.収穫胚および分析<ol><li>頸椎脱臼を経由してダムを生け贄に捧げる。簡単に母親から子宮角を除去するための開腹切開を展開します。子宮嚢の中の胚にしてください。チルドPBS中で胚を置きます。 </li><li> （あれば遺伝子型判定のために必要な収集組織）子宮嚢から胚を解剖し、全胚を置くI一晩30％ショ糖に一晩、その後nは4％パラホルムアルデヒド（PFA）、クライオスタット切片媒体中で凍結する。その後、凍結切片をカットし、免疫組織化学的分析のためにスライド上に置きます。必要に応じて、組織のために使用し、100％メタノール中で脱水し、4％PFAで固定に従って全体をマウント。 </li><li>免疫組織化学的分析のために10月を除去するためにPBSに凍結切片を置く。その後、30分間、10％正常ロバ血清でセクションをブロックする。 4℃で一晩100希釈：次に1でPECAM一次抗体を追加します。翌日、10分毎にPBTで3回スライドを洗浄し、二次ロバ抗ラット594を追加し、室温で1時間インキュベートする。 注：これは、超音波およびマイクロインジェクションの最適化と調整を必要とする非常に厳しい技術です。あり、この技術に関連した非常に急な学習曲線があり、1つが堪能になる前に指導し、注射の4〜6週間が必要です。 </li></ol>

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	<li>Abrahamson, D. R., Robert, B., Hyink, D. P., St John, P. L., Daniel, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+formation+of+microvasculature+in+the+developing+kidney.">Origins and formation of microvasculature in the developing kidney.</a> Kidney international. Supplement. 67, S7-S11 (1998).</li><li>Sims-Lucas, 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=Endothelial+Progenitors+Exist+within+the+Kidney+and+Lung+Mesenchyme.">Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme.</a> PloS one. 8 (6), e65993 (2013).</li><li>Dressler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</li><li>Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+controls+and+cellular+behaviors+in+branching+morphogenesis+of+the+renal+collecting+system.">Genetic controls and cellular behaviors in branching morphogenesis of the renal collecting system.</a> Wiley Interdiscip Rev Dev Biol. 1 (5), 693-713 (2012).</li><li>Costantini, F., Kopan , R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+a+complex+organ:+branching+morphogenesis+and+nephron+segmentation+in+kidney+development.">Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development.</a> Dev Cell. 18 (5), 698-712 (2010).</li><li>Das, 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=Stromal-epithelial+crosstalk+regulates+kidney+progenitor+cell+differentiation.">Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation.</a> Nat Cell Biol. 15 (9), 1035-1044 (2013).</li><li>Rymer, C., 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=Renal+blood+flow+and+oxygenation+drive+nephron+progenitor+differentiation.">Renal blood flow and oxygenation drive nephron progenitor differentiation.</a> Am J Physiol Renal Physiol. , (2014).</li><li>Shah, S. R., 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=Embryonic+mouse+blood+flow+and+oxygen+correlate+with+early+pancreatic+differentiation.">Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.</a> Developmental biology. 349 (2), 342-349 (2011).</li><li>Andres, A. C., 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=EphB4+receptor+tyrosine+kinase+transgenic+mice+develop+glomerulopathies+reminiscent+of+aglomerular+vascular+shunts.">EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.</a> Mech Dev. 120 (4), 511-516 (2003).</li><li>Wagner, R., 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=High-resolution+imaging+of+kidney+vascular+corrosion+casts+with+Nano-CT.">High-resolution imaging of kidney vascular corrosion casts with Nano-CT.</a> Microsc Microanal. 17 (2), 215-219 (2011).</li></ol>

The authors declare that they have no competing financial interests.

マイクロインジェクション麻酔と時間枠母親の麻酔に関しては、それは気流定数（2-3 L /分）と低PSIに保つことが不可欠である。鎮静剤の流れは、約1.75から2 L / minで保持されなければならない。同時に、注射が行われている時間枠を綿密に監視し、各ごみとのために制御されなければならない。各ごみための注射の手順は45分以下に抑えます。各胚が身体と関心のある器?...

胚発生中に2つの別々の、まだ同時、血管のプロセスが行われます。血管新生、血管が住宅内皮前駆細胞1,2から血管のデノボ形成であり、主要な既存の容器、および脈管形成、から成長するプロセスを。後者は、主にそれが存在しない場合に起こると考えられている間に、それぞれ、前者は、血流と同義である。 血管形成への同時、腎臓前駆細胞合成、増殖、および分化の循環的かつダイナミックなプロセスは、胎生9.5（E9.5）上で展開を開始します。この時点で、尿管芽（UB）は、周囲の後腎間葉（MM）に背側に侵入し、出産3まで続く。急速に後腎キャップ間充織を凝縮にUBの繰り返しの分岐は腎臓の機能ユニットの形成、ネフロンを開始します。 UBとnephのすべての新世代ロンは、古い世代は、それらが、その後、主に血管密度の高い環境内でさらに成熟と分化を受け、内側皮質と髄質の地域、にずれている。ドレスラーらによって証明されるように、図3に、この発生学的プロセスは、UBとMMとの間のクロストーク、および細胞外因子3-6無数のように、誘導性シグナル伝達により沈殿させる。開発膵臓と腎臓内の二つの最近調査し、細胞外の要因は、酸素緊張と血流7,8が含まれています。後者は、腎臓の発生に関連して以下でさらに詳細に説明する。 血流は、潜在的ネフロン前駆細胞の分化、並びにの他の器官形成プロセス、胚血流マッピングの精密で正確な方法で果たす役割の誘導を露出させるために不可欠である。 血流を測る別の方法は、ulの処方を含むtrasoundイメージングと樹脂が9,10を投げかける。結論的に、これらのモードは、本質的に同時に、血流との間の時間的及び空間的な並置を発表し、幹細胞の分化する能力に欠けていることが示されている。レジンキャストは、例えば、成体組織内の血管パターニングの有効なモデルを提供する、しかしそのような胚の時点と同様に未熟船、で、血管が肉眼的に未発達及び漏出性である。そのため、樹脂は、しばしば多孔質の、小さな血管内に保持することができないキャスト。 これらの見かけ上の障害のために、とりわけ、我々は超音波ガイド下腎臓発生の私たちの調査に生体内で心臓内胚トマトレクチン（TL）マイクロインジェクションに組み込むことを選んだ。この手順では、同期E11.5、E13.5、E15.5およびE17.5の時点でのマウス胚の左心室にTL溶液2.5μlので満たされたマイクロピペット取り付けられた針を案内するために超音波プローブを利用する。 E17針がより発展した胚を貫通するのに十分な強さではありませんように0.5は、最新の発達時代です。 このマイクロインジェクション法の利点は豊富である。超音波ガイド下マイクロインジェクションは、動物の鼓動する心臓への胚性左心室、ソリューションの受動的かつ制御追放、心臓や周辺組織への最小限のダメージ、との突然の心不全と死の回避内注射針の正確な位置決めが可能胚全身灌流の前に。 FITC標識TLを使用することで、すべての灌流し、血管系は、内皮頂端膜に沿ってマーカーを維持します。免疫組織化学と組み合わせて、PECAM（CD31、血小板内皮細胞接着分子）、および様々な他の血管マーカーを利用して、我々は明らかに灌流および非灌流血管を区別するだけでなく、周囲の組織の任意の異常な染色を特徴付けることができる。

<table><tbody><tr><td>DAPI</td><td>Sigma Aldrich</td><td>022M4004V</td><td>concentration 1:5,000</td></tr><tr><td>Pecam</td><td>BD Biosciences</td><td>553370</td><td>concentration 1:100</td></tr><tr><td>FITC-Tomato Lectin</td><td>Vector Laboratories</td><td>FL-1321</td><td>concentration 2.5 &amp;micro;l / embryo</td></tr><tr><td>Alexa Fluor-594 (Donkey Anti-Rat)</td><td>Jackson Immunoresearch</td><td>712-585-150</td><td>concentration 1:200</td></tr><tr><td>Microinjection Needle</td><td>Origio Mid Atlantic Devices</td><td>C060609</td><td></td></tr><tr><td>Mineral Oil</td><td>Fisher Scientific</td><td>BP26291</td><td></td></tr><tr><td>1 ml syringe</td><td>Fisher Scientific</td><td>03-377-20</td><td></td></tr><tr><td>Clay Blocks</td><td>Fisher Scientific</td><td>HR4-326</td><td></td></tr><tr><td>Surgical Tape</td><td>Fisher Scientific</td><td>18-999-380</td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>NC9763655</td><td></td></tr><tr><td>Hair Removal Product</td><td>Fisher Scientific</td><td>NC0132811</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science tools</td><td>14084-08</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science tools</td><td>11064-07</td><td></td></tr><tr><td>Surgical Marking Pen</td><td>Fine Science tools</td><td>18000-30</td><td></td></tr><tr><td>Right angle forceps (for hysterectomy)</td><td>Fine Science tools</td><td>11151-10</td><td></td></tr></tbody></table>

in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow

腎臓の血管（離れて糸球体からの）開発の形成と灌流が大幅に代役されている。脈管構造は、（主要な血管の外分岐される）は、血管形成および脈管形成（ 新生血管形成）を介して発展するにつれて、そのような生体内超音波イメージングの樹脂キャスト、および顕微解剖として灌流マッピング技術との間の密接な関係を実証するが限定されているこれら二つのプロセス及び胚内の腎臓の構造を開発。ここでは、腎臓の灌流の個体発生を測定するためにマウス胚に子宮内心臓超音波ガイド下FITC標識トマトレクチンマイクロインジェクションでの手順を説明します。トマトレクチン（TL）は、収穫胚および腎臓を通して灌流した。ネフロン前駆細胞、ネフロン構造、尿管上皮、および血管系：組織を含む様々な腎臓構造に共染色した。 E13.5から始まる大口径容器は、しかし、末梢灌流した船がunperfused残った。 E15.5およびE17.5では、小さな末梢血管だけでなく、糸球体は、灌流さになることを始めた。この実験的手法は、胚発生中の脈管構造と血流の役割を研究するために重要である。

血管形成は、腎臓の開発フローに先行 （腎臓を含む）胚組織の大部分はあっても初期胚の時点で、密な血管系（unperfusedと灌流さの両方）が含まれています。よりよいゲージに、我々は子宮内胎児の心臓内マイクロインジェクションでの方法を利用した開発腎臓内の血流を分析。 E17.5を通してE11.5で胚の心を識別するために、高解像度の超音波の使用により、および開腹?...

Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

マウス胚に子宮内 、心臓内トマトレクチン注射では腎血流量を測定する

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

in-utero-intra-cardiac-tomato-lectin-injections-on-mouse-embryos-to

Research

JoVE Journal

Developmental Biology

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

9.7K ビュー.  Children's Hospital of Pittsburgh of UPMC. This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

ビデオ: In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

医学

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

高周波超音波を用いて、マウス胚におけるコントラストイメージング

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

発生生物学

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

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

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

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

ネズミの胎児心エコー検査

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

非侵襲Mモード心エコー検査によるマウスでの心拍数の子宮内測定で

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

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 ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

子宮内開発のマウスの脳および脊髄の伝達の後の微細構造神経可塑性パラメーターの評価

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

落射蛍光マウント全体を用いたマウス胚発生中に心臓商工会議所発展の分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortopathy β-アミノプロピオニ トリルによる大動脈瘤と解離のマウスモデルでのマイクロ CT 定量分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

マウス胎児心臓の開発に細胞標識と注入

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

マウス胚に子宮内、心臓内トマトレクチン注射では腎血流量を測定する

要約

さらに動画を探す

この動画の章

のマウスモデル子宮内で移植

高周波超音波を用いて、マウス胚におけるコントラストイメージング

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

ネズミの胎児心エコー検査

非侵襲Mモード心エコー検査によるマウスでの心拍数の子宮内測定で

マウスモデルにおける静脈内投与と胎嚢内子宮内移植

子宮内開発のマウスの脳および脊髄の伝達の後の微細構造神経可塑性パラメーターの評価

落射蛍光マウント全体を用いたマウス胚発生中に心臓商工会議所発展の分析

Aortopathy β-アミノプロピオニトリルによる大動脈瘤と解離のマウスモデルでのマイクロ CT 定量分析

マウス胎児心臓の開発に細胞標識と注入

マウス胚に子宮内 、心臓内トマトレクチン注射では腎血流量を測定する

要約

さらに動画を探す

この動画の章

のマウスモデル子宮内で移植

高周波超音波を用いて、マウス胚におけるコントラストイメージング

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

ネズミの胎児心エコー検査

非侵襲Mモード心エコー検査によるマウスでの心拍数の子宮内測定で

マウス モデルにおける静脈内投与と胎嚢内子宮内移植

子宮内開発のマウスの脳および脊髄の伝達の後の微細構造神経可塑性パラメーターの評価

落射蛍光マウント全体を用いたマウス胚発生中に心臓商工会議所発展の分析

Aortopathy β-アミノプロピオニ トリルによる大動脈瘤と解離のマウスモデルでのマイクロ CT 定量分析

マウス胎児心臓の開発に細胞標識と注入

マウス胚に子宮内、心臓内トマトレクチン注射では腎血流量を測定する

マウスモデルにおける静脈内投与と胎嚢内子宮内移植

Aortopathy β-アミノプロピオニトリルによる大動脈瘤と解離のマウスモデルでのマイクロ CT 定量分析