我々 は非常に栄趙、杰李 Ying ヤンの協力をありがとうございます。

ケアと健康の国民の協会によって公開された実験動物の使用および機関動物ケアおよび使用委員会 (IACUC) によって承認された動物のプロトコル ガイドに従って行われたこれらの研究議定書の下で薬の Baylor の大学の数、4203。
 1。 超音波妊娠マウスの作製 1700 以降 <ol><li><ol><li>実績のある肥沃な男性とダムの場所を 交尾のタイミング をマウスに一晩開始。別のダムと男性 0700 時間 (h)、関係なく妊娠の正確な年代測定を確保するために膣にプラグがある場合 。
		注: 0.5 日と見なされる交尾後の朝のポストの coitum 定め (d.p.c.) です</li>。
	</ol></li> <li>超音波のための準備 <ol><li>シール麻酔誘導コンテナーで妊娠中のマウスを配置します</li>。
		<li>落ち着いたイソフルラン (2%) 吸入麻酔薬と酸素 (1 L/分) まで動物を失い立ち直り反射 (約 1-2 分のサイズに応じてマウスの)、(呼吸) 以外にも自発的な動きが観察されていません</li>。
		<li>にノーズコーンとイソフルレン (2%) 吸入麻酔薬と酸素 (1 L/分) で仰臥位で鎮静マウスを置く。動物に眼軟膏を適用 &#39; s 目</li>。
		<li>綿棒で腹部全体、膣、胸骨に脱毛クリームを適用し、外側を側面します</li>。
		<li>は、最大 3 分のままにクリームを許可する、クリームとティッシュで髪を取り外します。残りの髪は画像品質が低下、すべての髪が削除されることを確認します</li>。
	</ol></li></ol> 2。超音波ステージの準備 <ol><li>探触子をオンにします</li>。
	<li>トランスデューサーに 3-D モーター段階モーター ステージを添付します。
	<ol><li>3 D にプラグ モーター、3 D モーター ケーブルを背面パネルの 3 D モーター コネクタに接続します。3 D モーター システムをイメージング ステーションに接続する上にクイック リリース ポストを使用してマウント システムにアタッチとクイック リリース マウント トランスデューサー クランプを貼る下</li>。
	</ol></li> <li>探触子クランプにプローブを接続します</li></ol>。
 3。 妊娠マウスをイメージング開始 <ol><li>場所マウス監視プラットフォームで仰臥位。マウスは継続的にイソフルラン麻酔 (1.5-2.5%) の間を受け取ると鼻の円錐形を介して酸素 (1 L/分)。心拍数監視プラットフォーム上のパッドにすべての足をそっとテープします。
	<ol><li>上腹部超音波伝送ゲルの 1-2 の mL を適用します</li>。
	</ol></li> <li>手動モーター ステージを使用して下腹部に超音波プローブを置きます。
	<ol><li>膀胱は、膣の開口部に頭の方だけ液体で満たされた暗闇の円として表示されますを見つけます</li>。
		<li>膀胱が見つかったら、プローブを移動非常にゆっくりと頭の方妊娠中の子宮は、妊娠サイトで丸いエリアと円柱の形として表示されますを視覚化します。これはチェーンに真珠のように見えるとして記述することができます</li>。
		<li>したら 2 D イメージング、妊娠子宮が発見されました</li>。
	</ol></li></ol> 4。2 D 超音波イメージング (図 1) <ol><li>妊娠子宮を特定したら、膀胱に最も近い妊娠サイトから始まり、ゆっくりと順番には妊娠のサイトの数と場所を決定するため頭側移動します</li>。
	<li>腎臓、脾臓や肝臓が可視化プローブの位置を変更近い尾 (に近い膀胱) ユーザーがあまりにも頭の方まで行っていると</li>。
	<li>同じ方法で採卵をイメージします</li>。
	<li>超音波フレームは後の分析や測定のために保存する注入/妊娠サイトの中心部でときに画像をフリーズする 。
	注: それは冷凍して後で分析のために保存画像の 1 秒未満かかります</li>。
	手動でクリックしてマーカーとしてエコー エストロージェン反応を利用した <li>測定注入距離、&#34; メジャー &#34; まず、ツールし、1 つの注入のサイトの場所をクリックします。次に、着床しコンピューターが自動的にその距離をレポート ラインを描画するをクリック、次にカーソルをドラッグします。ソフトウェアは、手動測定が報告が。この手順はないコンピューターですが、プログラムは測定先決を提供 decidualized サイト間の線で decidualized のサイト間の距離をマークするユーザーに依存している</li>
	<li>注入、胎嚢サイズ、胎児極サイズを測定します</li>。
</ol> 5。胎児心拍 注: 9.5 d.p.c. で胎児の心臓の鼓動を明確に視覚化する必要があります。
<ol><li>プローブを非常にまだ維持しながらパルス波ドップラーをオンにし、表示のハートビートにこれを置きます</li>。
	<li>レコード、心臓鼓動脈動 。
	注: 中心部から離れてこの手順も記録できます脈動に臍帯</li>。
</ol> 6。 3-D 超音波買収 <ol><li>後、妊娠中の子宮は 2 D 画像の可視化されて目的の 3-D イメージのおおよその中間点にある領域にプローブを位置づけ。たとえば、3-D でリンゴを撮像された 1 つ場合プローブされるべきである最初する位置 (すなわちオブジェクトの真ん中) をリンゴの芯が予測されています</li>。
	<li>の目的のイメージの中にプローブを用いた 3-D 取得を取得します。3 D モーター ステージは一連の手順またはフレームのために子宮全体でこの距離を旅行、どのような調査官の欲望全体に取り込むつもりで</li>。
	<li>意図されていた構造がリアルタイム超音波部分を完了する前に 3 D スキャンで完全にキャプチャされたことを確認します。後でポスト処理のためこの 3次元の情報を保存します。2 D と 3 D の超音波取得の所要時間は約 10-20 分経験のあるユーザーによって実行されるときです</li>。
</ol> 7 3次元再構成後処理 (図 2) <ol><li>3次元画像処理の必要なデータをロードします。</li>。
	<li>選択 &#34; 平行・回転メソッド &#34;、すべての 3 D 画像のフレーム 1 つの 3-D に負荷が &#34; ボックス &#34; ユーザーは、フレーム単位での目的のオブジェクトをトレースし。ステップを選択サイズ 0.08 mL。画像ブロックの一方の端から始まり、空間内で撮影されている画像を理解するスクロールします。
	<ol><li>イメージと手動でトレース オブジェクトのアウトラインの一方の端で開始します</li>。
		<li>次の 2 D イメージ スライスをスクロールまたは &#34; フレーム &#34; オブジェクトのアウトラインを手動でトレースと</li>。
		<li>手動で注釈/トレース ユーザーによってすべてのフレームがされるまで、このプロセスを続行します</li>。
		<li>をクリックして &#34; を終了 &#34; 3次元画像と総体積の計算を取得する</li>。
	</ol></li> <li>メッシュと左上隅の矢印は、固体のオーバーレイを選択します</li>。
	<li>を保持または削除する周辺の超音波イメージ情報をわかりやすくするために選択します。それぞれの 3次元復元が 10 〜 20 分間かかることがあります</li>
</ol> 8。手続き後ケア <ol><li>超音波が完了したら、麻酔ガスをオフに、プラットフォームから、マウスを削除しふきます、動物から、超音波ジェルを洗い流します</li>。
	<li>は、パディング領域の腹臥位でケージに戻ってマウスを配置します。動物は自然に目を覚まし、移動するまで監視します</li>。
</ol>

<ol>
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著者が明らかに何もありません。

3次元 HFUS イメージングのこの小説の使用は、正常に検出、視覚化、および非侵襲的方法で妊娠初期マウス胚着床を特徴付ける機能を示します。技術は、総組織と病理組織学的評価のために妊娠の中断に依存する現在の方法と比べて大幅に改善を提供しています。しかし、組織学的方法まだと思われるより最適なより拡大しより細胞レベルで評価が必要なときや、遺伝子・ タンパク質解析が?...

再発妊娠初期の損失は受胎後は最も一般的な合併症の 1 つと1,2を想像しようとするカップルの約 1% に影響を与えます。妊娠初期の損失のメカニズムは変わる: 本質的な胚の異常や子宮内膜の感受性1,3,4の欠陥に母体合併症から。自分の遺伝的少ないのためのマウス モデルは、初期胚移植の妊娠の調査のため広く利用されています。さらに、マウスと大規模な研究を実行する能力の短い妊娠時は、人間の生殖医学5の主要な臨床質問に対処でマウスの成長しているユーティリティを確保しています。つまり、マウスの実験的デザインの大半はまだ定量化妊娠6、中に注入サイトの場所、数、サイズ、および間隔のパターンを分析して順次妊娠日の安楽死させたことに多数のダムを必要とします。 7、8、それによって同じ動物に関する縦断的研究は除外。
クリニックで超音波は人間の胎児の生存、非侵襲的方法9,10,11の開発を監視する信頼性の高い、非常に貴重なツールです。最近では、高周波超音波 (HFUS) は、マウスの胎児の生存および妊娠12,13,14中の成長を監視する方法として限られたアプリケーションを検索し始めています。超音波画像における最近の技術の進歩は、動物の臓器の視覚的な復元と病態15,16、それ以降の監視の立体 (3 D) データのアプリケーションを許可しています。 17。この高度なイメージング技術の使用より小さいボリュームの変動、動物間の可変性を減らすために、病理学の進行または17の治療的介入の有効性を監視するための検出力は飛躍的に。この技術の主要なユーティリティは、営利的モデル15,16における悪性腫瘍の進行を監視してきましたが、3-d HFUS ばかり量的に表わすし、胚移植の積極的な成長を監視する使用されていますマウス子宮18で胎児の発育。
ここでは、初期の妊娠マウス子宮の再構成を生成する 2 D と 3 D データを生成するイメージング HFUS を実行する方法を示します。非侵襲的な方法でデータを収集するために研究者を許可する妊娠の終了を必要とせずこれらの初期胚の注入のイベントを検出するこの手法の有用性を示す.

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			<td>VisualSonics Vevo 2100 Ultrasound Imaging Platform/Machine</td>
			<td>VisualSonics, inc.</td>
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			<td>Vevo Imaging Station</td>
			<td>VisualSonics, inc.</td>
			<td>SA-11982</td>
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			<td>Aquasonic 100 Ultrasound Transmission Gel</td>
			<td>Parker</td>
			<td>#SKU PLI 01-08</td>
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			<td>Isoflurane (IsoThesia) 100mL bottle</td>
			<td>Henry Shein</td>
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			<td>PuraLubenAnimal Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>#12920060</td>
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a novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse

高周波超音波 (HFUS) は、非侵襲的子宮内胎児のリアルタイムの開発を監視するための一般的な方法です。マウスは体内モデルとして胚移植と妊娠の進行の勉強を日常的に使用されます。残念ながら、このようなマウスの研究は、フォロー アップの表現型解析を有効にする妊娠の中断を必要とします。この問題に対処するため立体 (3 D) HFUS 画像マウス胚の注入のサイトと、個々 の発達進行で子宮の早期検出と評価のためのデータを使用しました。我々 は、正確に胚移植サイト数を定量化すると同様に 9.5 d.p.c. を通じて 5.5 日ポスト膣 (d.p.c.) から C57BL6J/129S 妊娠マウスにおける発達の進行を監視ができた HFUS 3次元再構成画像とモデルを組み合わせて、探触子を用いる。含まれている測定: 数、場所、およびサイト間注入間隔と同様、着床のボリューム胚の生存率は、心臓の活動を監視することによって評価されました。すぐ着床後期間 (5.5 〜 8.5 d.p.c.) 再構成メッシュと固体のオーバーレイの両方で妊娠の子宮の形式にはそれぞれの子宮角内にある発展途上の妊娠の視覚的表現が有効になります。遺伝子改変マウスが子宮の機能不全から派生した女性の生殖形質を特徴付けるために使用するのにつれ、このメソッドは、検出、定量化、および初期注入イベント特徴づける体内への新しいアプローチを提供しています。3次元 HFUS イメージングのこの小説の使用は、正常に検出、視覚化、および非侵襲的方法で妊娠初期マウス胚着床を特徴付ける機能を示します。技術は、総組織と病理組織学的評価のために妊娠の中断に依存する現在の方法と比べて大幅に改善を提供しています。ここでメッシュと立体画像で子宮の形の改築に伴う信頼性と再現性のあるデータを生成するのにマウス妊娠初期の超音波を正常に実行するのに方法について説明ビデオ、テキスト形式を使用します。

図 1に示されているように、高周波超音波は注入サイト開発、早くも 5.5 着実にポイントを検出できます。ライターのエコーを使用して 6.5 着実に着床のマーカーとして decidualized 子宮内膜は着床と間隔を定量化するこれらのサイトの数をことができます。妊娠として 7.5 d.p.c.、暗い低エコーの胎嚢と胎仔のポールまで進行は簡単に識別もできます...

Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

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

マウスは、妊娠性生物を勉強する広く使用されます。ただし、妊娠の終了は、縦断的な調査を排除する、多数の動物の使用を必要とするこのような研究に必要です。したがって、早期発見のための高周波超音波の非侵襲的手法と妊娠マウスに着床後イベントの監視について述べる。

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 ...

a-novel-use-three-dimensional-high-frequency-ultrasonography-for

Research

JoVE Journal

Developmental Biology

8.1K ビュー.  Baylor College of Medicine. マウスは、妊娠性生物を勉強する広く使用されます。ただし、妊娠の終了は、縦断的な調査を排除する、多数の動物の使用を必要とするこのような研究に必要です。したがって、早期発見のための高周波超音波の非侵襲的手法と妊娠マウスに着床後イベントの監視について述べる。

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

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

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

マウス子宮内妊娠中の母体の血管リモデリングの評価

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平全マウント：皮膚の厚い3次元組織断面のための新規処理および画像化プロトコール

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

三次元レンダリングとカルビンディン抗体、解析人間の胎盤絨毛血管のネットワークを明らかにしました。

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

歯肉の炎症性三次元人体同等

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

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

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

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

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

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

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

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

前庭と聴覚の感覚器官の三次元切片培養

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

マウス膵臓前駆細胞の in Vitroからスキャフォールド フリー、三次元のインスリンを表現する Pancreatoids の生成

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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 ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

初期マウス胚からのシーケンシングのための小型RNAライブラリーの調製

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...