その技術支援、はなフレードとスザンヌ シュルツに感謝します。本研究は資金を受けてないです。

このプロトコルは、動物実験 LANUV レックリングハウゼン (#84-02.04.2016.A374) の倫理委員会によって承認されました。成体マウスでこの手順を実行する一般的には、> 10 週齢。しかし、若い動物と同様にこの手術を行うことが可能です。手術器具は、使用前に滅菌する必要があります、すべてのステップが無菌条件の下で実行されます。
1. 麻酔の挿管誘導
<ol><li>痛みを軽減するため腹腔内ブプレノルフィン 0.1 μ g/g の体重を注入します。手術後の次の 3 日にブプレノルフィン 0.1 mg/kg 8 時間毎の投与を繰り返します。</li>
	<li>誘導、イソフルランの 3.0 の巻 %1 L/分の酸素流量の設定気化器に接続されている麻酔誘導ボックスにマウスを配置します。</li>
	<li>触覚刺激を誘導することによって深い麻酔を確認します。 メモ: イソフルランの増加 Vol % まで 5%、麻酔導入に失敗した場合は麻酔は十分に深くないです。</li>
	<li>反射がないことを確保するのには、マウスの尾をピンチします。反射の完全な不在は、最適な人工呼吸器設定のマウスの重量を量る (1.12 を参照)。</li>
	<li>プロシージャ全体で 37 ° C の体温を維持するために温度制御オペレーティング テーブルにマウスを移動します。</li>
	<li>麻酔の維持麻酔誘導ボックスに接続されているプラスチック製のコーンに、マウスの鼻を配置します。</li>
	<li>ナイロン縫合糸で、マウスの上顎を固定します。粘着テープで手足を固定します。</li>
	<li>十分な麻酔を再び確保する鉗子の先端で後肢に圧力を適用します。逃避反射の不在で、次の手順に進みます。 注: は麻酔導入が失敗するか、麻酔は十分に深くない場合イソフルランの容積 % を増加し、逃避反射の不在を待ちます。</li>
	<li>麻酔下での乾燥を防ぐために角膜に滅菌眼科用潤滑剤を配置します。</li>
	<li>直腸の外傷を避けるため直腸プローブを塗ります。37 ° C の体温を確保するための直腸温度プローブを挿入します。</li>
	<li>喉と胸の上部と製造元の指示に従って脱毛クリームを depilate します。1 分後、クリームを拭き取る。必要な場合は、成功するまでこの手順を繰り返します。 注: は、出血した場合は綿棒を使用します。</li>
	<li>70% のエタノールの depilated 領域を清掃します。ポビドン ヨードは、皮膚消毒 3 回、少なくとも 3 分を適用します。</li>
	<li>生理学的パラメーターに人工呼吸器の設定を調整します。150/分と 8-10 μ L/g 体重 (BW) 換気量に呼吸速度を設定します。</li>
	<li>滅菌手袋の新しいペアに置きます。手術顕微鏡下にマウスを置き、マウスより滅菌穴開きドレープを配置します。</li>
	<li>正中 2ndリブまで下顎で約 3 mm の皮膚を切開します。正中線や顎下腺の結合組織を識別します。関節包内鉗子を使用して優しく 2 つの鉗子で露骨に正中線で腺を分割し、気管筋を探索します。</li>
	<li>パラ気管の筋肉を関節包内鉗子で露骨に離れて引っ張って気管をゆっくり準備します。</li>
	<li>簡単に挿管条件の喉をまっすぐにし、気管内挿管カニューレ (外径 1.2 mm) をそっと挿入鉗子で舌を引っ張ってください。気管内チューブの直接可視化し、適切な胸の動きをチェックして、挿管を確認します。</li>
	<li>1.0 L/分、100% O2の流れと 2% イソフルランに対する挿管後イソフルレンの濃度を調整します。 注: 呼吸運動が停止しない場合、マウス移動を開始最初 180/min まで呼吸数を増加します。必要に応じて、濃度を高めるイソフルラン 3.5% 第二マウスが独自に呼吸を停止するまで。最も一般的な問題として、漏出または気化器の不十分な詰物を評価します。</li>
	<li>また、次の sub 手順で推奨されている気管挿管を行います。<ol>
<li>60 ° の角度でテーブルの上のマウスの位置。</li>
		<li>固執粘着テープでマウスの四肢と頭部のリクライニングします。</li>
		<li>喉頭の上肌に直接冷光光源を配置します。</li>
		<li>声帯を視覚化するための鉗子で、舌を優しく引き出します。</li>
		<li>声帯を介して IV 型カニューレ (24 G) のプラスチック製のチューブを挿入し、人工呼吸器設定にプラスチック製のチューブを接続します。</li>
		<li>シンクロン胸の動きによって挿管を確認するためカニューレに人工呼吸器を接続します。 注: 呼吸運動が停止しない場合、マウス移動を開始最初毎分 180 まで呼吸数を増加します。必要に応じて、濃度を高める二次イソフルラン 3.5% までマウスが独自に呼吸を停止するまで。最も可能性の高い問題として漏出または気化器の不十分な詰物を評価します。</li>
	</ol>
</li>
</ol>2. 術前計測法
<ol><li>鉗子で結合組織を軽く引き離すによって気管を置く補助の両方の頸動脈を準備します。</li>
	<li>左右 45 ° より小さい角度で左内頚動脈にいくつかの滅菌された超音波ゲル 20 MHz ドップラー プローブの先端を配置します。</li>
	<li>外側と内側に検索しドップラー信号を信号を最適化するためにプローブを傾斜移動するプローブをゆっくりと回転させます。</li>
	<li>ドップラーのソフトウェアを使用して表示し、右と左総頚動脈コンピューター上で流速を格納します。</li>
</ol>3. 開
<ol><li>手術部位感染を防ぐためにそれぞれの個々 のマウスの滅菌手袋のセットを使用します。</li>
	<li>ハサミで 2nd肋下切開を展開します。</li>
	<li>肋骨を数えることで、2ndの肋間スペースを視覚的に識別および関節包内鉗子でこのスペースをにべもなく浸透します。 注: 1st肋骨、鎖骨の下にある、したがって表示されません最初目に見えるリブ (すなわち2ndリブ) と 3rdリブ間 2nd肋間スペースを見つけたので。</li>
	<li>鉗子のヒントの助けを借りて 2nd肋間スペースを開き、リトラクターを挿入します。</li>
	<li>胸腺の明確な見解を持っているオペレーティング テーブルに接続されているゴムバンドで、リトラクターを調整します。</li>
	<li>出血の場合綿の先端を使用し、2 分の表在性の血管に押します。</li>
</ol>4. 横方向の大動脈の縞模様
<ol><li>正中線や結合組織を識別するために 200% に倍率を調整します。鉗子を使って優しく胸腺を分割します。大動脈弓は明らかに見ることができるまでは、脂肪組織を削除します。</li>
	<li>斜め下 (図 1参照) 腕頭動脈と左総頚動脈間横断の大動脈鉗子を結ぶトンネルを準備します。微細鉗子の助けを借りて 6.0 縫合糸の端を保持し、大動脈のアーチの下のスレッドを渡します。</li>
	<li>アーチの反対側から 2 番目の鉗子でスレッドを取る。</li>
	<li>27 G の針を使用する長さは 3 mm 長い部分を切断、マウス マウス用スペーサーとして 19 25 g 体重 26 G 針と計量用 TAC 結紮用スペーサー > 25 g BW。</li>
	<li>横方向の大動脈に平行スペーサーを慎重に配置します。</li>
	<li>スペーサーを緩い二重結び目を準備し、大動脈に平行にスペーサーの最適な配置を確保します。最初のスローを結び、すぐに 2 番目の逆スローを実行します。スペーサーを速やかに削除します。</li>
	<li>コントロールで偽マウスを実行するには、大動脈結紮を省略する同じプロトコルに従います。</li>
	<li>6.0 ポリプロピレン縫合糸で 2nd肋を閉じます。縛ること鎖骨下動静脈に特別な注意を払います。</li>
	<li>連続縫合パターンで 6.0 ポリプロピレン縫合糸を使用して皮膚を縫合します。</li>
</ol>5. 横大動脈結紮の確認
<ol><li>セクション 2 のように 45 ° の角度で首の両側に 20 MHz ドップラー プローブを配置します。</li>
	<li>それぞれの側に流速を文書化します。 注:図 2に示すように、成功した TAC はドップラー流速によって検証できます。左右の内頚動脈は通常間 4 - 10 フロー速度比により十分な TAC (図 2参照)。</li>
</ol>6. 心の収穫
<ol><li>1.2 の手順に従って酔いを誘発します。1.3。</li>
	<li>安楽死室にマウスを置き、ボリューム/分の 10-30% を転置する炭酸ガスの流量を調整します。</li>
	<li>手術テーブル上でマウスを固定します。詳しく分析するためカニューレと下大静脈からハサミと収穫の血と腹部を開きます。</li>
	<li>強力なハサミで、横隔膜と胸骨の骨をカットし、中心部を削除します。</li>
	<li>心を計量する前に、すべての動脈と結合組織を削除します。</li>
	<li>右心室と左心室の中隔を分離し、両方のサンプルの重量を量る。</li>
	<li>両方の組織サンプルを液体窒素で凍結します。</li>
</ol>

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著者が明らかに何もありません。

TAC による高血圧の急激な発症は、大動脈弁狭窄症や高血圧によって引き起こされる臨床的に関連の肥大によって異なります。それにもかかわらず、心不全を誘発する小さな動物モデルの使用は多くの利点し、は、したがって、多くの調査官11によって選ばれています。この閉胸モデル マウス4で横方向の大動脈狭窄を誘導する手技の既存モデルが向上しま?...

マウス モデルは人間の病気1を模倣するために使われます。圧負荷を誘発する横方向の大動脈狭窄 (TAC) を使用し、左心室肥大2。ロックマンらによって検証されたマウスにおける開胸タック モデル3外科的処置は DeAlmeidaらによって、詳しく説明します。4します。 横方向の大動脈のバンディングは腹部大動脈狭窄と比較してより有利な循環のより大きい部分は、この後者の手順2の負の影響を補うことができますので。
昇順大動脈および腕頭動脈で、増加する動脈圧につながる大動脈横のバンディングが、(すなわち左総頸動脈、左鎖骨遠位血管を介して臓器の十分な血流を葉動脈および大動脈)。これは向上心後負荷と昇格した心臓壁のストレスに します。壁応力は、その後繊維肥厚5による減少します。慢性心臓血行動態変化は不適応と左の心室の膨張の結果します。こうすると、TAC は、最終的に心不全につながる心臓肥大の再現モデルを作成します。
DeAlmeideらによる記述で TAC の標準的な手順4肋骨または胸骨と縦隔と胸腔に入る郭清による部分上部開胸による大動脈弓に近づきます。大動脈弓とその側枝の良い表示が可能になります。残念なことに、切り裂かれた肋骨を再アタッチできません、それらの葉が浮遊していると、呼吸のダイナミクスを変更すること。
、我々 はしたがって、2nd肋間間隙を介して横外科的アプローチを使用して大動脈弓へ低侵襲閉じた胸アプローチを確立しました。このモデルの最大の利点は、肋骨を介しても切断せずに TAC を実行する機能です。周手術期は皮膚の切開と肋間筋の解剖に限定されます。このプロシージャは自体外傷を最小限に抑える、十分な胸の安定性を維持するのに役立ちます。
ここで我々 はマウスの合計または上部開胸を行わず TAC 手術を実行する詳細な手順をについて説明します。高周波ドップラーは、TAC の成功は前述した6,7を確保するためだった。

<table border="0" cellpadding="0" cellspacing="0" width="1303">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:312px;">Pressure-volume catheter</td>
			<td style="width:243px;">Millar Instruments, USA</td>
			<td style="width:591px;">SPR-839</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse ventilator</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>Minivent - TYPE 845</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse ventilator</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>Y-connection with intubation cannula OD 1.2mm 73-2844</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vaporizer</td>
			<td>Dr&#228;ger Medical AG&#38;CO.KG, Germany</td>
			<td>19.3 Isofluran-Vaporizer (a newer version is available under catalog number&#160; D-877-2010)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope&#160;</td>
			<td>Leica Microsystems, Germany</td>
			<td>MZ 7.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light source&#160;</td>
			<td>Schott AG, Germany</td>
			<td>KL 1500 LCD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-0 Prolene</td>
			<td>Ethicon, USA</td>
			<td>Polypropylene suture BV-1 9.3 mm 3/8c</td>
			<td>suture for surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Seraflex</td>
			<td>Serag Wiessner, Germany</td>
			<td>USP 5/0 schwarz;&#160; IC108000&#160;</td>
			<td>suture for constriction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Homoeothermic Controlled Operating Tables</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>Typ 872/3 HT with tripod stand and homoeothermic controller Type 874; 73-4233</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flexible Rectal Probe</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>1.6 mm OD; 55-7021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Doppler Signal Visualisation Instrument</td>
			<td>Indus Instruments, USA</td>
			<td>Doppler Signal Processing Workstation (DSWP) with 20MHz Pulsed Doppler Module</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doppler Probe</td>
			<td>Indus Instruments, USA</td>
			<td>20MHz Tubing-mounted Probe</td>
			<td></td>
		</tr>
	</tbody>
</table>

a closed-chest model to induce transverse aortic constriction in mice

心肥大・心不全研究頻繁、TAC による圧力オーバー ロード マウス モデルに基づいています。標準的な手順は、横方向の大動脈弓を視覚化する部分的な開胸手術を行うことです。ただし、開胸モデルにおける開胸手術による外科的外傷は、肋骨を切除し、左開胸後未接続呼吸生理を変更します。これを防ぐためには、側方開胸による低侵襲、閉胸アプローチを設立しました。ここに取り組んで大動脈弓 2nd肋間間隙を介して胸部キャビティを入力せず少ない外傷から回復すると、マウスを残しています。等しい生存率で開胸タック プロシージャの標準的な実験室の設定を使用してこの操作を行います。閉胸アプローチのための生理学的な呼吸パターンを維持し、離れてマウスは外傷後の免疫反応を減らすために高速治癒過程を容易にする表示されます低侵襲技術として急速な回復を示すことによって恩恵を受けるようです。

成功した TAC は、圧負荷、左室肥大の誘導を保証します。圧負荷のアドホック検証は、図 2に示すように、ドップラー流速測定を使用して実現できます。術前の血流速度は両方の頸動脈に等しいが、TAC は、拡張血速度左心室および大動脈圧の上昇により右頚動脈後狭窄弱毒血流速度を起こしながら、左頚動脈。
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Watch this Scientific Journal Video about A Closed-chest Model to Induce Transverse Aortic Constriction in Mice at JoVE.com

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

ここでは、側方開胸による横方向の大動脈狭窄 (TAC) のプロトコルを提案します。この手法は、圧負荷および TAC 研究室の標準設定を利用したマウスの心臓障害をシミュレートすることを目指して低侵襲、閉胸手術です。

マウスで横方向の大動脈狭窄を誘導するために閉じて胸モデル

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

a-closed-chest-model-to-induce-transverse-aortic-constriction-in-mice

Research

JoVE Journal

Medicine

18.8K ビュー.  University Hospital Bonn. ここでは、側方開胸による横方向の大動脈狭窄 (TAC) のプロトコルを提案します。この手法は、圧負荷および TAC 研究室の標準設定を利用したマウスの心臓障害をシミュレートすることを目指して低侵襲、閉胸手術です。

ビデオ: A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

CYP2D6の動物モデル：マウスにおける自己免疫性肝炎を誘導する方法

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

医学

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

マウスを用いた気管狭窄のin vitroでの測定で

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

心筋虚血再灌流障害のマウスクローズド胸モデル

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

圧負荷心肥大モデルの作成のためのラットの大動脈狭窄昇順

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

従来のクローズド·チェスト法による心室細動と蘇生のラットモデル

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

磁気マイクロビーズ閉塞モデルマウスにおける緑内障高眼圧症依存誘導すること

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

心臓形態学的および心エコーイメージングによって横大動脈狭窄のマウスモデルにおける機能の変更の評価

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

マウスにおける低侵襲横大動脈狭窄

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

左室肥大の誘導マウスにおける低侵襲の横大動脈狭窄のテクニック

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

組織と血液サンプリング手順の標準化による閉胸ブタ心筋梗塞モデルの改善

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...