We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1.組織の収穫と筋組織の凍結<ol><li>イソフルラン（2-クロロ-2-（ジフルオロメトキシ）-1,1,1-トリフルオロエタン）の過剰摂取で、​​マウスを安楽死させます。ヒュームフードで、このステップを実行し、そのようなデシケーターやイソフルランに浸した綿棒を含むベルジャー等の二次封じ込めメソッドを使用します。 </li><li>しっかりとフットパッドを絞ることによって死を確認してください。このような頸椎脱臼などの安楽死の二次メソッドを使用します。 </li><li>筋肉の緩み髪の毛の付着を低減するために、70％のアルコールで毛を濡らします。このような＃5のように微細な鉗子を使用して、四肢切断、皮膚の皮をむきます。 </li><li>慎重に、下骨から（この場合は前脛骨筋（TA））、目的の筋肉を分離腱から開始して削除します。 </li><li> 、筋肉を切除10月（最適切断温度化合物）でそれをカバーし、標識された使い捨ての凍結金型の上に平らに横たわっていました。筋肉は、その正常な生理的姿勢であることを確認してください（TAIにヘッド延伸することなくL）。あるいは、小さな発泡スチロールの正方形の筋肉を凍結し、筋肉の正しい長さを確保するために昆虫ピンを使用してピン。 </li><li>液体窒素を用いた鋼ビーカーに2-メチルブタン（イソペンタン）を冷やします。 5分 - これは、平均3になります。白い沈殿物をビーカーの底と壁に形成し始めるまで、断続的に攪拌してください。 注：この場合、イソペンタン、最適な凍結温度に到達した（-150°C） </li><li>チルド2-メチルブタンの筋肉を含む金型を浸し。 12秒と腓腹筋ヒラメ筋（GS）と15上腕三頭筋などの大きな筋肉 - - 20秒かかる6ダイアフラムと長指伸筋（EDL）などの小さな筋肉をフリーズします。あまりにも長い間、この亀裂につながる可能性としてのための筋肉を凍結しないでください。 </li><li>ドライアイスの上で凍結組織を移し、（このプロセスは15の周りの平均をとることに注意してください - 20分）イソペンタンを蒸発させて。アルミ箔で組織をラップとuでそれらを格納切片までLTRA低温（-70℃〜-80℃）。 </li></ol> 2.組織ブロックを準備するために10月に組織を埋め込みます<ol><li>ドライアイス上で輸送組織が融解を防ぐためにcyrostatします。 （組織の完全融解を回避するために、C°-24、-20に設定）チャンバをクライオスタットし、それらを30分間平衡化させるために組織を転送します。 </li><li>の冷却ペルチェベイの電源を入れます。クライオスタットは、この冷却を持っていない場合は、すぐに10月を凍結するエアロゾル冷却スプレーを使用しています。このような埋め込み中の任意の時点での筋融解は、凍結損傷を与えることができないようにしてください。 </li><li>試料ディスク上の10月の均一な薄層を追加し、冷まします。上にまだ液体ながら10月のほとんどが底に凍結されている場合には、（横断面と縦断のための10月に平行のため10月に垂直な方向）正しい向きで組織を挿入します。すぐにOCTを凍結するエアロゾル冷却スプレーを使用してください。熱EXTと組織のunembeded部分に触れてractor、45秒間待ちます。 </li><li> 、筋肉の周りに10月の別の薄い層を追加し、エアロゾル冷却スプレーで噴霧し、前のステップと熱を抽出。 </li><li>少しずつで10月を追加し続けると、すぐに全体の筋肉がカバーされるまでエアロゾル冷却スプレーおよび熱抽出器の組み合わせを使用して、10月にフリーズ。 </li><li>組織ブロックの上に熱抽出を入れ、5分間待ちます。 </li></ol> 3.切片を作製し、筋肉のミッド腹領域からのセクションを識別する<ol><li>試料ヘッドの上に試料をマウントします。ディスクを右に回して、締め付けます。試料ディスクを試料ヘッドとの良好な接触状態にあることを確認します。 </li><li> °-21の間および-24℃に室内温度を調整し、設定温度が達成されるまで待ちます。 </li><li>厚さの設定（7μm）を調整します。粗微動の設定を使用してブロックを前進または後退させることにより、ブロックをトリミング。 </li><li>セクションを収集上のスライドに切断面の付着を容易にするために、反対の電荷間の引力を使用して、セクションの上にスライドの上部を押して、正に荷電した顕微鏡スライドを予熱しました。 </li><li>セクションの断面積（​​CSA）を測定することにより、ミッドベリーセクションを識別します。顕微鏡に接続されているコンピュータにこの使用してイメージングソフトウェアを実行してください。 注：ソフトウェアプログラムのトレース機能を介して位相コントラスト画像が取得され、筋肉全体の円周を測定します。これは、CSA、ならびに最小フェレット直径（粒子の反対側にある2つの平行な接線の間の最小距離として表現さ​​れる繊維断面サイズの測定）測定の両方が得られます。注：CSAと最小フェレット直径測定値がプラトーに開始すると、半ば腹に達しました。 </li><li>将来の染色のために-80℃でスライドをインベントリおよびアーカイブします。 注：10月前staininに削除する必要はありません。G手順。スライドをカバーガラスをされると、彼らはすぐに染色される準備ができています。 </li></ol> 4.染色注：詳細なステップバイステップの手順は、製品データシートに記載されていますし、染色手順のために従うべきであるが、個人の最適化は、最適な染色を得るために必要とされ得ます。 <ol><li>メディアは（表を参照）の取付は、水と混和しないので、（70％、95％、および100％エタノールに2分間ずつ）アルコールの増加グレードを通してスライドを脱水し、キシレン（5分間、100％）を確保するためにそれらをクリアスライドは、時間をかけて不透明になることはありません。 </li><li>セクションをカバースリップし、それらが乾燥してみましょう。イメージングソフトウェアを備えた顕微鏡を用いた画像。 </li></ol>

<ol>
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N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy.">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33, 845-853 (1985).</li><li>Maccarty, W. 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The authors have nothing to disclose.

組織学および免疫組織化学は、様々な神経筋障害で病状の症状および進行を理解する上で重要なツールとして使用されています。古典的には、筋肉生検は、最初のホルムアルデヒドで固定し、その後パラフィンワックスで包埋しました。ホルムアルデヒド固定は、組織構造を保持し、室温で組織保存および輸送を可能にするが、それはまた、正確な免疫染色​​を達成するためのさらなるス?...

筋肉組織学の検討は、神経筋障害1-4の異なるタイプの理解に重要な役割を果たしています。他の技術と組み合わせると、それは研究者が病状の症状および進行のより良いアイデアを得ることができ、また、治療的介入5-10の有効性を評価するために不可欠であることができます。しかし、正確に、組織を直ちに切除前の生理的状態を維持するように適切な方法で保存する必要があります。これは、除去、保存、切片、および染色の際に細心の注意が必要です。 組織を光学顕微鏡研究のため、2つの異なる方法で調製する​​ことができます。 （FFPE）のセクション、埋め込 ​​まれた第1の方法、ホルマリン固定パラフィンは、組織はパラフィンワックス11,12に埋め込 ​​まれる前に、10％の自然緩衝ホルムアルデヒド中に固定することが必要です。固定工程は、より良い形態を維持するのに役立つだけでなく、クロスリンク内因性のPRoteinsは、このように免疫染色のための複雑な抗原回復手順を必要とし、このようなセクション12の酵素染色の可能性を排除します。凍結切片を、一方、予め固定または処理する必要はありません。したがって、タンパク質は、天然の生物学的な立体配座13,14を保持することができます。また、凍結切片はまた、時間に肉腫および他の外科的生検15の術中診断で、たとえば、必要である、迅速なターンアラウンドタイムを可能にします。適切に行われていない場合は、この方法は、組織学的検査のためのセクションでは、不向き作る筋肉のアーキテクチャの変更を紹介しダメージを、凍結する傾向があります。本稿では、適切な検査のために最適な染色を達成するために、筋肉生検を準備するための最も効果的な方法を概説します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Olympus</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Nikon Imaging Software Basic Research</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>M 32631-SL</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
	</tbody>
</table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

筋肉生検の組織学的評価は、神経筋疾患の病態の発症および進行の理解に不可欠なツールを務めています。しかし、そうするためには、適切なケアは、最適な染色を達成するための組織を切除し、保存するときに注意が必要です。組織保存する一つの方法は、ホルムアルデヒドで組織を固定し、その後パラフィンワックスでそれらを埋め込むことを含みます。この方法は、よく形態を保持し、室温で長期保存を可能にしますが、面倒であり、有害な化学物質の取り扱いが必要です。また、効果的な免疫染色​​のための抗原回復プロトコルを必要と抗原架橋、ホルムアルデヒド固定をもたらします。逆に、凍結切片は、固定を必要とし、したがって、生物学的な抗原の立体構造を保持しません。この方法は、intraoperaような迅速な組織学的評価を必要とする状況で、それは特に有用なもの、時間の周りクイックターンで明確な利点を提供します外科的生検を的。ここでは、異なる組織学的および免疫学的汚れによる可視化のための筋肉生検を調製する最も効果的な方法を説明します。

収穫および組織の凍結のためのセットアップを図1に見ることができる。切除した組織を乾燥する（ 図1A）を回避するために、PBSに浸したガーゼ上に格納されるべきです。組織を組織学的分析のために使用されるように選択される場合、それらは、OCT中に浸漬し、適切な向きでクライオモールド上に配置し、正確な形態（ 図1B）を確保することができるよ...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do&#039;s and Don&#039;ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

組織学的分析のために筋肉凍結切片の調製でのやっていいことといけないことを行います

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

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Medicine

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

31.4K ビュー.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

ビデオ: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

ビボ犬筋肉の機能アッセイに

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

医学

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

ハイスループットフローサイトメトリー細胞ベースのアッセイN-メチル-D-アスパラギン酸受容体またはヒト血清中のドーパ​​ミン-2受容体に対する抗体を検出する

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

マウスの圧負荷モデルを用いた冠動脈の流れと冠血流準備の超音波ベースのアセスメント

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人間<em&gt;外側広筋</emワイル・ブレークスリーConchotomeを使用&gt;骨格筋生検

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

マウスの膵臓の組織学的解析と代謝プロファイリングに対する郭清

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

マウスの心臓形態の心エコー検査・組織学的検討

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

ラット舌の機械的および粘弾性的特性のインビボ評価

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

メタボロミクス解析とリピドミクス解析のための脳腫瘍の現場でのサンプリングと抽出

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

循環器疾患大型動物モデルにおける心臓の解剖技術と組織学的サンプリング

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

長期培養のためのヒト心筋組織の作製

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...