Name: fetal mouse cardiovascular imaging using a high-frequency ultrasound (30/45mhz) system
Uploaded: 2018-05-05T12:30:00.000+00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System at JoVE.com

テクニカル サポートと Vevo 2100 超音波生体顕微鏡 (UBM) システムへのオープン アクセスを提供するため ucla 医学部動物生理学コアに感謝いたします。この研究に支えられた今日と明日こども基金は、カリフォルニア大学ロサンゼルス校-子供のディスカバリー研究所 NIH/小児保健研究センター (5K12HD034610/K12) と m. 当麻にデイヴィッド ・ ゲフィン医学学校の研究イノベーション賞を受賞。

このプロトコルに示すようにすべてのプロシージャを承認したは、カリフォルニア大学、ロサンゼルス、動物のケアおよび使用委員会。[アクティブな動物プロトコル制度動物ケアおよび使用委員会のカリフォルニア大学、ロスアンジェルス、カリフォルニア、米国によって承認された進行中の研究の一環として実験を行った。動物の処理とケアは、ケアと実験動物の使用のためガイドの基準を続いた。
1. 高周波超音波撮像システムの準備
<ol><li>超音波イメージング システムと生理学の監視ユニットを入れます。</li>
	<li>30/45 MHz の探触子を接続します。</li>
	<li>イメージング プラットフォーム近くそのホルダーに対応するスキャン ヘッドを配置します。</li>
	<li>心臓の測定プログラムオプションを選択します。</li>
	<li>超音波ゲル逆さまの 37 ° C に設定済み温暖化コンテナー内の場所</li>
	<li>麻酔の適切なチューブ システムを確認し、酸素とイソフルランのレベルを確認します。</li>
	<li>イメージング プラットフォームと作業領域を消毒します。</li>
	<li>一定の体温を維持するために画像処理プラットフォームの熱レベルとダムの心拍数を設定します。</li>
</ol>2. 妊娠マウスの作製
<ol><li>麻酔誘導室で妊娠マウス (C57/BL6) ダムの場所。</li>
	<li>継続的に 100% 酸素 (100% O2) 誘導室 200 mL/min の流量で混合吸入イソフルラン (イソフルラン 2%-3%) を配信を使用して麻酔を誘導します。</li>
	<li>仰臥位でイメージング プラットフォームに鎮静の動物を転送します。</li>
	<li>定常状態の鎮静麻酔チューブ システム提供イソフルラン (1.0%-1.5%) に接続されているフェイス マスクを使用して混合 100 %200 mL/分で O2を提供します。 注意: は、キャニスター セットを含む木炭フィルター搭載換気システムを用いた麻酔ガスの漏洩を制御します。</li>
	<li>母体の心臓と呼吸率の常時監視を達成するために電極のゲルの塗布後埋め込み心電図電極に優しく手足をテープします。</li>
	<li>(450 ± 50 拍/分 (bpm)) の平均心拍数を維持するためにイソフルランのレベルを調整します。</li>
	<li>± 0.5 ° C 37.0 ° C の範囲内で体温を維持します。体温と生理コント ローラー ユニットに表示される心拍数を監視します。</li>
	<li>鎮静マウス イメージング プロシージャ全体で 15 分毎のバイタル サインを文書化します。</li>
	<li>マウスの姿勢、心拍数、つまみをつま先への応答を評価することによって麻酔のレベルを評価します。</li>
	<li>目の乾燥や角膜の損傷を防ぐために眼クリーム (それぞれの目に 1 滴) を適用します。</li>
	<li>超音波減衰を最小限に抑えるために脱毛クリームを使用して下肢に半ば胸のレベルからの毛皮を削除します。ウェットとドライのガーゼを交互にアプリケーションが皮膚に損傷を防ぐためワイプ後クリーム 1 〜 1.5 分間を削除します。</li>
</ol>3. 胚識別
<ol><li>胎児を検索しそれらを広げて優しく腹壁を触診します。</li>
	<li>ダムの腹部にそれぞれの胚に注釈を付けるし、マーカーを使用して、その前後と背腹軸方向を定義します。</li>
	<li>ランドマークとして鎮静のダムの子宮頸部を使用します。L1, L2, L 3 として左と右子宮角に胎児をラベル等(左側) と R1、R2、R3、等(右側にある)、それぞれ (図 1 a)。 注意: は、胎児を強制的に感染を避けるため。各くずの 1-2 胎児は、他の信頼性の低い自分のポジショニングとイメージングを作ると重なる可能性があります。これらの胎児を分析から除外します。</li>
</ol>4. 胎児心臓の可視化と注釈
<ol><li>腹部に予め温めておいた超音波ゲルを適用し、気泡を避けるために注意深くそれを広めます。スキャン画像の領域に追加のジェルの量を追加します。</li>
	<li>その機械のホルダーに超音波プローブを配置し、スキャン B モード (図 1) を使用して心臓の鼓動を探しながら厚いゲル層と接触する皮膚に向かって徐々 にそれを動員します。</li>
	<li>2 D イメージを取得するスキャンB モードボタンをクリックします。ランドマークとして、膀胱を使用して右または左子宮角に配置最初胎児を識別し、それぞれ R1 か L1 としてそれをマークします。</li>
	<li>リアルタイムで個々 の胎児の左右の向きを確認するには、水平面内イメージング プラットフォームを移動します。(図 1 bビデオ 1) のランドマークとしての鼻、手足と背骨に注釈を付けるための尾に頭からスキャンします。</li>
	<li>心臓の鼓動を視覚化し、左心室 (LV)、右心室 (RV) に注釈を付けます。心の可視化を最適化するために使用色ドップラー モード (図 1 C Gビデオ 1-2)。</li>
	<li>旁短い軸のビューを取得するスキャンB モードボタンをクリックして LV と RV データ集録フレームの中央に、最大径表示があります。ライブ イメージング (図 1 b・ C) を開始します。</li>
	<li>縦 4 部屋ビュー (図 1) を取得する平面をスキャンに関して、マウスの方向を変更します。まず、心房、心室中隔、左と右の流出管など心の残りの構造を識別します。次に、その最大径表示心室と心房のチャンバーを持っています。画像取得を開始します。</li>
	<li>最終的な分析から最適ではない、斜めの画像を除外します。最低 10 の連続記録 'Cineloops' を取得するチーニボタンをクリックして s、記録された画像を保存します。</li>
</ol>5. 胎児の心拍数と心機能の評価
<ol><li>4 室内機 (動画 3) から心臓の画像を取得するスキャンM モードボタンをクリックします。</li>
	<li>すべての胚の画像が完了したら、解析のための録音の一覧を表示します。</li>
	<li>最終的な分析から最適ではない、斜めの画像を除外します。</li>
	<li>壁厚を測定し、拡張期 (LVID、d 項の権限で右/左心室内径分析ボタンをクリックします。RVID、d) と収縮 (LVID、s;RVID、s)、図 2に示すように。</li>
	<li>それぞれを再生することにより平均胎児心拍数記録 M モード トレース、次のフローを 1 つのフロー サイクルの測定の計算サイクル (隣接したピーク間の間隔) を決定します。</li>
	<li>平均心拍数 (図 2) を取得する複数の測定値 (少なくともトレースあたり 5) を実行します。</li>
	<li>左心室内部拡張期径 (LVID、d) と心臓サイクル時間 (LVID, s) で左心室内径の変化を測定します。小数短縮率 (FS %) を次のように計算: FS % = [(LVID,d-LVID,s)/LVID、d] x100。</li>
	<li>FS % の平均値を取得する複数の測定値 (少なくともトレースあたり 5) を実行します。</li>
</ol>6. 心肺フロー パラメーターの評価
<ol><li>60 未満o買収の角度でセクターを調整します。ドップラーボタンで 45 MHz の探触子を使用して、2 D 4 商工会議所イメージング平面からパルス波ドップラー測定を実行するをクリックします。<ol>
<li>まず、右の流出路を識別するために肺動脈の分岐を視覚化します。次に、パルス波ドップラーボタン (図 3 aビデオ 4) 大動脈弁、肺およびを介してフロー パターンを取得するをクリックします。</li>
	</ol>
</li>
	<li>パルス波ドップラー トレース、収縮期波 (PkV)、(AT) の加速時間駆出時間 (ET) などからの肺血流測定を取得します。</li>
	<li>(右)図 3 aに示すように平均測定値を取得する複数の測定値 (少なくともトレースあたり 5) を実行します。</li>
	<li>AT を計算/各エ比流出管開存性の指標としてバルブを流出し、血流。</li>
	<li>パルス波ドップラーを使用して 2 D 頂 4 室ビューから僧帽弁と大動脈弁の流れパターンの取得に進んでください。まず、左心房と左心室のチャンバーを識別します。次に、(A) (図 3 b)24,25僧帽弁流入ドプラ パターンと測定拡張早期波 (E) と心房収縮速度の記録するためパルス波ドップラー サンプル ボリュームを配置します。</li>
	<li>大動脈のドップラー ジェット パターンを取得するドップラー サンプル ボリュームを調整します。大動脈ドップラー jet (右)図 3 bに示すように、(で) 時間と駆出時間 (ET) の加速度を測定するため (動画 5) を使用します。</li>
</ol>7. 胎盤の軸の評価
<ol><li>45 MHz 探触子 (図 4 a) を使用して、子宮動脈および胎児胎盤血管ツリーを視覚化色ドップラー スキャンを使用します。</li>
	<li>胎児の腹部からコードが終了する直後に臍帯、羊水内のセグメントの膀帯血管 (2 つの動脈と静脈の 1 つ) を識別します。</li>
	<li>場所パルス波ドップラー サンプル ボリューム臍動脈の流れパターン (図 4 a) を取得します。</li>
	<li>(AT)、駆出時間 (ET)、加速時間を含む血管ピーク フロー パラメーターを測定し、ピークの時間 (PkV, s) の流速パルス波ドップラー スキャン レコード (図 4 b) を使用します。</li>
	<li>胎動や母体の呼吸運動、各容器の平均ピーク速度を測定するの不在で、各船で 5 連続波形を取得します。</li>
	<li>次の胚に進みます。</li>
</ol>8. イメージ作成後動物の監視
<ol><li>イメージング プロセスの完了に続くイソフルラン コンテナーをオフにします。</li>
	<li>回復期の体温、呼吸数、心拍数の監視を続けます。</li>
	<li>ダムが自発的な動きを始めたら、フェイス マスクと接続チューブ システムを削除します。</li>
	<li>適切なハウジングにダムを戻り、標準機関ポスト プロシージャ プロトコルに従って観察を続けます。</li>
	<li>通常の活動の完全復旧に時間を文書化します。</li>
</ol>9. パフォーマンス要件および技術的な考慮事項
<ol><li>バイタル サインの生理学的パラメーターの長時間の麻酔の副作用を避けるために約 1 h 〜 8 胎児の処理の時間を制限します。</li>
	<li>画像集録およびフロー パターンの短いタイム フレームでトレースの技術を最適化するために 8-10 妊娠マウス トレーニングを完了します。</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Folate-Related+Gene+Environment+Interactions+and+Congenital+Heart+Defects.">Maternal Folate-Related Gene Environment Interactions and Congenital Heart Defects.</a> Obstet Gynecol. 116 (2 Pt 1), 316-322 (2016).</li><li>Gabbay-Benziv, 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=A+step-wise+approach+for+analysis+of+the+mouse+embryonic+heart+using+17.6+Tesla+MRI.">A step-wise approach for analysis of the mouse embryonic heart using 17.6 Tesla MRI.</a> Magn Reson Imaging. 35, 46-53 (2017).</li><li>Kim, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+fetal+echocardiography.">Murine fetal echocardiography.</a> J Vis Exp. (72), e4416 (2013).</li><li>Zhou, Y. Q., Cahill, L. S., Wong, M. D., Seed, M., Macgowan, C. K., Sled, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+flow+distribution+in+the+mouse+fetal+circulation+at+late+gestation+by+high-frequency+Doppler+ultrasound.">Assessment of flow distribution in the mouse fetal circulation at late gestation by high-frequency Doppler ultrasound.</a> Physiol Genomics. 46 (16), 602-614 (2014).</li><li>Greco, A., Coda, A. R., Albanese, S., Ragucci, M., Liuzzi, R., Auletta, L., Gargiulo, S., Lamagna, F., Salvatore, M., Mancini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Frequency+Ultrasound+for+the+Study+of+Early+Mouse+Embryonic+Cardiovascular+System.">High-Frequency Ultrasound for the Study of Early Mouse Embryonic Cardiovascular System.</a> Reprod Sci. 22 (12), 1649-1655 (2015).</li><li>Deneke, T., Lawo, T., von Dryander, S., Grewe, P. H., Germing, A., Gorr, E., Hubben, P., Mugge, A., Shin, D. I., Lemke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+determination+of+the+optimized+atrioventricular+delay+in+patients+with+implanted+biventricular+pacing+devices.">Non-invasive determination of the optimized atrioventricular delay in patients with implanted biventricular pacing devices.</a> Indian Pacing Electrophysiol J. 10 (2), 73-85 (2010).</li><li>Kono, M., Kisanuki, A., Ueya, N., Kubota, K., Kuwahara, E., Takasaki, K., Yuasa, T., Mizukami, N., Miyata, M., Tei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+global+systolic+dysfunction+has+a+significant+role+in+the+development+of+diastolic+heart+failure+in+patients+with+systemic+hypertension.">Left ventricular global systolic dysfunction has a significant role in the development of diastolic heart failure in patients with systemic hypertension.</a> Hypertens Res. 33 (11), 1167-1173 (2010).</li></ol>

利害の対立が宣言されていません。

心血管系奇形や疾患は、遺伝的要因と環境要素19に影響されて大幅に。我々 は以前、胎児胎盤循環流と胎児心機能92 番目の妊娠中に開始、母体のカロリー制限の大きな影響を実証しました。
出生前の低酸素は、途方もなく胎盤の生理学、循環系に影響を与えるかもしれない胎児の発育中にもう一つ一般的なストレス要因です。出生前?...

心臓の先天異常が早期の心臓の開発中に発生する異種の構造上の欠陥です。運用手順の現在の技術の進歩は、CHDs1,2児の生存率の大幅な改善につながっています。しかし、生活の質はしばしば長期入院やニーズに侵害された二次手術手順1,2,3,4,5を上演します。出生前診断 CHDs の分子機構の解明は、新たな予防戦略を遂行し、生涯の成果6,7を改善する早期介入を計画するために重要です。
CHDs 病態では、複数の遺伝子と環境要因が関与しているが診断、治療、改善するために満たされていない必要性と予防戦略1,8,9 のまま因果関係を確立します。 ,,1011,12。さらに、子宮内でのストレス要因とエピジェネティックな修飾子の役割を調べて将来調査11,12の新しい会場を開きます。最後の十年は確かに次世代シーケンス技術単一ヌクレオチド多型 (SNP) マイクロ アレイ、全エキソーム配列、ゲノム広いメチル化研究など遺伝の勉強に利用の急速な進歩を目撃しています。CHDs1,8,9,10,11を新規変異とされていない遺伝的変異を識別するために道を舗装を含む複雑な人間の病気の原因します。適切な動物モデルにおける病原性のテスト。
別の病気モデル システム間でマウスは13,の心臓初期14,15,16時でも解明する CHDs のメカニズムを調査するだけでなく、好みの動物モデル心臓チャンバー成熟と出生前および周産期のストレス要因の胎生後期に機能への影響。したがって、生体内で表現型特徴づけを行う変異マウス胎児心臓の開発の初期段階および後期段階ではこれらの遺伝的変異と心臓の開発の環境要因の役割を理解することが重要とマウスにおける商工会議所特定の成熟プロセスの潜在的な将来の影響。
早期発見と開発の間に心臓障害の正確な診断は、介入計画17,18にとって重要です。セーフ、シンプル、ポータブルで反復可能であること、胎児超音波画像クリニックで心臓の評価のための技術標準となっている確かに。超音波ドプラを用いた胎児循環評価が広く使用されて臨床心臓欠陥の検出のためだけでなくも血管異常、胎盤機能不全や子宮内胎児発育制限を検出し、評価するには胎児の福利子宮内低酸素血症、母体の病気、薬物毒性17,18などの侮辱への応答。人間の欠陥と疾患の評価に値に並行して、超音波検査胎児マウスの実験設定19,20,21,22、増加ユーティリティを得ています 23。特に、胎児心臓超音波 (心エコー) は、心臓の開発の連続した生体内で可視化できます。多くの実験的研究は、トランスジェニック マウス胎児の胎児心血管を観察するのに超音波イメージング技術を使用しています。ドップラー超音波は、生理学的課題や病気の条件10,19の下で胎児循環のフロー パターンや、病態生理学的パラメーターを明らかにする特に役立っています。人間と動物の両方で胎児への異常な血液の流れや酸素供給に起因することができます子宮内胎児環境破壊および胎盤軸、胎盤異常を含む母体低酸素症に影響を与えるさまざまな条件妊娠糖尿病と薬学的誘導血管狭窄15,22.したがって、胎児マウスのドップラー超音波を実行するための標準化された方法の確立途方もなく権限を与える CHDs の将来の研究中に心血管回路のキーの血行動態指標と監視フロー パターンを促進することで遺伝的マウスモデル.の心臓の開発のさまざまな段階
高周波超音波は、マウス モデルおよびひと疾患18で心臓血管系の発達的および生理学的パラメーターを測定するための強力なツールとして浮上しています。この技術は、ここ数年でさらに洗練されてきた。我々 と他の研究者は、胎児マウス中心15,19,20,21,22 の超高周波超音波研究のためこのシステムの有効性を実証しています。 ,23。色ドップラー フロー マッピング、リニアアレイ探触子高周波 (30 ~ 50 MHz) のフレーム レートで二次元でダイナミックな画像を生成するシステムが備わります。これらの利点は、低周波超音波診断装置と高周波超音波21,22, 前の世代に比べて循環、胎児の詳細な評価のため必要な感度と分解能を提供します。システムは、実験の設定で心構造、商工会議所の関数、および胎児マウスのフロー指標の包括的な評価を含みます。ここで、我々 は高周波装置を使用して心肺循環と萌芽期日 E18.5体内で胎児胎盤循環の迅速な評価を実行する方法を概説します。約 60 μ m の軸解像度および 150 μ m の空間分解能を提供する 30/45 MHz の探触子を選びました。ただし、次のような方法論的アプローチによって以前の発達段階を分析する高周波数探触子 (40/50 MHz) を選択できます。選択した M モードでは、高時間分解能のレベル (1,000 フレーム/秒) での運動の組織の描出が可能します。最後に、我々 は胎児心臓血管の血行動態と機能およびベースラインでマウス出生前の低酸素ストレス応答の詳細な包括的な表現型特性の高い超音波の可能性を実証します。

<table><tbody><tr><td>Vevo 2100</td><td>VisualSonics, Toronto, Ontario, Canada</td><td>N/A</td><td>High Freequency Ultrasound Biomicroscopy. The set up is available in animal physiology core facility, division of molecular medicine, UCLA. USA</td></tr><tr><td>inbred mice (c57/BL6)</td><td>Charles River Laboratories</td><td>N/A</td><td>Inbread wild type mouse strain</td></tr></tbody></table>

fetal mouse cardiovascular imaging using a high-frequency ultrasound (30/45mhz) system

先天性心疾患 (CHDs) は、子供の罹患率と早期死亡の最も一般的な原因です。出生前診断 CHDs の分子機構の解明は、新しい予防と治療戦略を発明にとって重要です。変異マウスのモデルは、新しいメカニズムおよび CHDs に心臓の開発とその潜在的な変質をドライブ環境ストレス修飾子を発見するための強力なツールです。これらの推定貢献者の因果関係を確立する努力がされている非生存動物実験組織学的・分子学限られた主要な生理学的および血行動態的パラメーターを監視するには、しばしば欠席。ライブ イメージング技術 CHDs の病因を確立する重要なツールとなっています。特に、超音波画像診断は外科的に胎児をさらすことがなく出生前使用できる商工会議所心臓の血行動態と構造面の環境ストレスの影響を監視しながら自分のベースラインの生理機能を維持することができます開発。ここで、我々 は心血管システム胎児マウス E18.5子宮内のベースラインに、出生前の低酸素暴露に応答を調べる高周波超音波 (30/45) システムを使用します。我々 は心臓チャンバー サイズ、形態、心機能、胎児の心拍数と臍動脈流インデックス、および全身の慢性的な低酸素症子宮内で実にさらされている胎児マウスで彼らの変化を測定するシステムの可能性を実証します。時間。

オフライン心・血行動態の指標の統計的解析を行った。3 つの最適な画像で 5 連続測定の手段を求めた。データは ± SEM. スチューデントのtの意味と表現された-テストは、集団間比較を推論に使用されました。0.05 のP値が統計的に有意と考えられました。
上記プロトコルに従って我々 はで C57/BL6 タイミング妊...

Watch this Scientific Journal Video about Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System at JoVE.com

高周波超音波 30/45MHZ システムを使用したマウス胎児心血管イメージング |発生生物学 |ビデオ

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

心血管イメージング (30/45 MHZ) の高周波超音波を用いた胎児マウス

高周波超音波胎児マウスの解像度の画像が上がったし、心臓発生と構造欠陥の正確な非侵襲的評価を提供することができます。本プロトコルを実行するリアルタイムの胎児マウス エコー生体内で設計されています。

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

fetal-mouse-cardiovascular-imaging-using-high-frequency-ultrasound

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Developmental Biology

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Human Biology

Circulatory and Pulmonary Systems

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11.5K ビュー.  University of California, Los Angeles. 高周波超音波胎児マウスの解像度の画像が上がったし、心臓発生と構造欠陥の正確な非侵襲的評価を提供することができます。本プロトコルを実行するリアルタイムの胎児マウス エコー生体内で設計されています。

ビデオ: Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

マウス心エコー検査と超音波イメージング

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

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

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 ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

マウスの早い妊娠の特性の三次元の高周波超音波の新規な使用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

胎児心エコー検査と子宮内発育制限のウサギモデルにおけるパルス波ドップラー超音波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

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 ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

体内の胎児と胎盤の開発の分析のための高周波超音波

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

私のマウスは妊娠していますか?高周波超音波評価

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

High-frequency Ultrasound Imaging of the Abdominal Aorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and <a href="https://www-jove-com.bdigitaluss.remotexs.co/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
High-frequency ultrasound systems are used to acquire high resolution images. Here, the use of a state-of-the-art system will be demonstrated to image the morphology and hemodynamics of small pulsatile arteries and veins found in mice and rats. Ultrasound is a relatively inexpensive, portable, and versatile method for the noninvasive assessment of vessels in humans as well as large and small animals. These are several key advantages that ultraound offers compared to other techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and near-infrared fluorescence tomography (NIRF). CT requires ionizing radiation and MRI can be prohibitively expensive and even impractical in some scenarios. NIRF, on the other hand, is limited by the penetration depth of light required to excite the fluorescent contrast agents.
Ultrasound has limitations in terms of imaging depth; however, this may be overcome by sacrificing resolution and using a lower frequency transducer. Abdominal gas and excess body weight can severely diminish image quality. In the first case, the propagation of sound waves is limited, while in the latter case, they are attenuated by overlying tissues, such as fat and connective tissue. As a result, no contrast or faint contrast may be observed. Finally, ultrasound is a highly user-dependent technique, requiring the sonographer to be familiar with anatomy and to be able to work around issues, such as the appearance of imaging artifacts or acoustic interference.

High-frequency Ultrasound Imaging and Image Acquisition

腹部大オルタの高周波超音波イメージング

Source: Amelia R. Adelsperger, Evan H. Phillips, and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, 
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