著者はアンドレアアクステル聡伊藤、MD、ジェフVelotta、MD、グラントホイト、ロバートC.ロビンス、MD、金玉、M​​D、ティムドイル博士、および技術支援のためのスタンフォード大学小動物のイメージングコアを感謝。また、作者は獣医機器のサポートのためにAMビックフォード社に感謝。この研究は国立衛生研究所（R01 HL - 75774、R01 CA098303、R21 HL085743、1K12 HL087746）、カリフォルニア大学のカリフォルニア州のたばこ関連病研究プログラム（15IT - 0257と1514RT - 0169）からの研究補助金によって支えられて、および再生医療（RS1 - 00183）のためのカリフォルニア工科大学。 NHは、アメリカ心臓協会からのフェローシップでサポートされています。

1。片側後脚虚血の誘導<ol><li>この操作に必要な外科的なツールが含まれます：細かい尖ったピンセット、鉗子、春のはさみ、外科はさみ、針ホルダー、およびリトラクターを指摘した。それは市販のリトラクターよりも小さいので、我々はペーパークリップを使って私たち自身のリトラクタを行います。オートクレーブまたはホットビーズ滅菌器で、手術前にこれらのツールを滅菌する。焼灼器ツールと滅菌細かい尖った綿棒も、この手術のために必要となります。それは手順の実行中に必要に応じてツールをヒントに再滅菌することをお勧めします。 </li><li>ツールの準備が整うと、1L/minの流量で100％酸素でイソフルラン1〜3％を含む麻酔導入チャンバーにマウスを置きます。 </li><li>それは外的刺激に反応しなくなるまで誘導チャンバ内でマウスを残す。その後、誘導室から動物を削除します。それはイソフルランの演算子の曝露を減らすために、蓋を開ける前ボックスから麻酔薬をフラッシュすることをお勧めします。 </li><li>その後、事前に手術台に仰臥位に動物を配置し、イソフルランの連続的な流れに接続します。電気シェーバーを使用して、後肢から毛を取り除く。徹底的に髪を削除するには、脱毛クリームを適用します。 </li><li>手術台の上にドレープ加熱パッド上に仰臥位にマウスを置き、イソフルランの連続的な流れに接続します。テープの切れ端で後肢を拡張して固定します。後肢が安全になると、三交互betadineとアルコールスクラブで皮膚を拭いてください。外科的処置の残りの部分では、後肢地域の拡大図を得るために10倍または20倍の倍率で解剖顕微鏡を使用してください。 </li><li>細かい鉗子と外科はさみを使用して、大腿内側に向かって膝から、約1 cmの長さ、皮膚の切開を行います。 </li><li>リン酸緩衝生理食塩水を使用して（PBS）、微細な先の尖った綿棒を湿らせ軽く太ももの筋肉を周囲の皮下脂肪組織を拭う。 </li><li>次に、切り込みを入れると、基礎となる大腿動脈を明らかにするために皮下脂肪組織を解剖して横方向に焼灼を適用します。 </li><li>傷口を開くことと、下肢の血管の良い眺めを得るためにリトラクターを使用してください。 </li><li>神経血管束を公開する膜大腿骨シースを通して微細な鉗子と細かい尖った綿棒、静かにピアスを使用する。 </li><li>その後、細かい鉗子や綿棒のクリーンセットを使用して、脚の付け根に近い近位の場所で大腿静脈と神経から大腿動脈を摘出し、分離する。解剖した後、大腿動脈の近位端の下に7〜0絹縫合糸の鎖を渡します。ダブルノットを使用して近位大腿動脈を閉塞。第二のタイとtransectedされ、間にセグメントの長さを残すために、できるだけ傷の近位等の容器にネクタイを置きます。 </li><li>膝に近い遠位の​​位置で大腿静脈から大腿動脈を分離。膝窩動脈の近位大腿動脈の遠位端の下に7〜0縫合糸の鎖を渡します。ダブルノットを使用して血管を閉塞させる。 </li><li>ノットの最初のセットにちょうど近重結び目の2番目のセットと遠位大腿動脈を閉塞。縫合糸のこの番目のセットは、トランザクションの手順中に動脈を把持するために使用されます。 </li><li>同様に、グリップの目的のために、結び目の最初のセットにちょうど遠位二重結び目の2番目のセットとの近位大腿動脈を閉塞。 </li><li>細かい尖った綿棒と春のはさみと遠位と近位の結び目の間に大腿動脈のセグメントを横断する。ピアス大腿静脈の壁はない、十分に注意してください。 </li><li>リトラクターを削除し、5から0 Vicryl縫合糸を使用して切開を閉じてください。これらの縫合糸は、彼らが自分自身で解散と、後で削除する必要はありません。 </li><li>切開がクローズされると、回復のケージで覆われた加熱パッドの上に動物を配置し、目を覚ましになるまで継続的に監視する。 </li><li>動物は1時間回復した後は、虚血の誘導を確認するためにレーザードップラー血流のステップに進みます。 </li></ol> 2。レーザードップラー血灌流<ol><li>レーザードップラー血流のステップを開始するには、1L/minの流量で100％酸素でイソフルラン1〜3％を含む麻酔導入チャンバーにマウスを置きます。 </li><li>それは外的刺激に反応しなくなるまで誘導チャンバ内でマウスを残す。その後、誘導室から動物を削除します。それはイソフルランの演算子の曝露を減らすために、蓋を開ける前ボックスから麻酔薬をフラッシュすることをお勧めします。 </li><li>イソフルランの連続的な流れに接続してプレオペレーティングテーブルの上に動物を配置。その後、必要に応じて脱毛クリームに続いて、電気シェーバーを使用して、後肢から毛を取り除く。 </li><li>毛を除去した後、イソフルランの連続的な流れの下で5分間、37℃で加熱された表面に動物を置く。温度変化が著しく血流に影響を与えるとして、euthermiaを確保するためのコア温度を監視します。 </li><li> 5分後、イソフルランの連続的な流れに接続されているような緑色の布のような非反射光吸収面、上に仰臥位に動物を配置。後肢を延長する。 </li><li>次に、レーザードップラーイメージャと買収のソフトウェアをオンにし、ソフトウェアを初期化する。ビューと分解能のフィールドのサイズを指定します。それは、将来の分析を容易にするために動物の間のビューと画素密度のフィールドの一貫性を保つのが最善の方法です。 </li><li>新しいファイルを開きます。画像データの取得を開始するSTART（スタート）を押します。一般的に、イメージャは自動的にマウスの距離を検出しますが、要求された場合は、イメージャへの動物の距離を指定します。レーザーは、ビューのフィールドの境界線を表示し、データの取得が開始されます。 </li><li>買収が完了すると、画像は、足への血液灌流のレベルの指標である色の範囲を示すために開始されます。色はよりよい動物の間でデータを比較するために特定の灌流の範囲に設定することができます。 </li><li>データ収集が完了したら、ファイルを保存します。 </li><li>その後回復のケージに動物を返し、目を覚ましになるまで継続的に動物を監視する。 </li><li>データを分析するために、あらゆるバックグラウンドノイズを減算する解析ソフトウェアのしきい値の調整を使用してください。 0.2のしきい値は、通常、妥当な値です。各後肢領域をカバー2関心領域、またはROIを、選択してください。ランドマークの様々な手足と動物の間でROIを標準化するために使用することができます。その後、ROI内の平均流と変動性を決定する。虚血肢と対照肢との間の血流の差と血流比は、容易に決定することができます。 </li><li>この手順は、時間をかけて後肢灌流の変化を追跡するために繰り返すことができます。希望する時点で、動物は、組織の機能と血流の画像データとの比較の評価のために安楽死することができます。 </li></ol> 3。代表的な結果： 後肢血管系の解剖学的構造を図1 2に示されています。大腿動脈の植後の後肢の代表的な図を図2に示します。図3に示すように、後肢の虚血の誘導を確認するには、レーザードップラー血流の画像解析は、対照肢と比較して、虚血肢への血流の劇的な減少を示しています。 <img src="/files/ftp_upload/old/1035_2009_1_26_9/figure1_1035.png" alt="図1" width="600" height="406" /> 図1。後肢の血管系の解剖学。アスタリスクは、後肢虚血の誘発のためのライゲーションの位置を示している。 <img src="/files/ftp_upload/old/1035_2009_1_20_7/Niiyama_HLI_Fig2.png" alt="イメージ2" width="600" height="450" /> 図2。大腿動脈を除去した後の近位および遠位部位における大腿動脈の結紮後の後肢の解剖学的構造を示す代表図。 <img src="/files/ftp_upload/old/1035_2009_1_20_8/Niiyama_HLI_Fig3.png" alt="図3" width="600" height="256" /> （3）レーザードップラー画像が左後肢（矢印で示される）に虚血の誘導前後での血流を示す図 。

<ol>
	<li>Huang, N. F., Niiyama, H., De, A., Cooke, J. P., , . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+non-invasive+tracking+of+embryonic+stem+cell-derived+endothelial+cells+for+treatment+of+hindlimb+ischemia.">Transplantation and non-invasive tracking of embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia.</a> J Vis Exp. , (2008).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The anatomy of the laboratory mouse. , (1976).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Dokun, A. O., Keum, S., Hazarika, S., Li, Y., Lamonte, G. M., Wheeler, F., Marchuk, D. A., Annex, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+trait+locus+(LSq-1)+on+mouse+chromosome+7+is+linked+to+the+absence+of+tissue+loss+after+surgical+hindlimb+ischemia.">A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.</a> Circulation. 117, 1207-1215 (2008).</li></ol>

後肢虚血モデルを計画し、実行中に考慮すべき変動性のあるソースがあります。最初に、虚血のレベルでは、側枝のことに関してライゲーションの場所に応じて異なる場合があります。したがって、モデルの一貫性を保つために、動物は動脈結紮の同じレベルを受けなければならない。虚血性回復にばらつきの別のソースが古い動物（8-10ヶ月齢）より速く、より完全な回復率を有する若い動物（6-8週齢）で、動物の年齢に関連している、などの血行動態（つまりによって評価レーザードップラー血流）や機能的な（すなわち、トレッドミル試験）対策。群間差が大きく、治療介入で観察されることがありますので、一つは血管新生阻害剤を評価されている研究については、それは、古い動物を使用することが好ましいかもしれない。逆に、一つの抗血管新生因子を評価されている研究のために、それは効果の大きさ3を最大化するために、若い動物を使用することが好ましいかもしれない。 正しく実行した場合、出血、感染、または死亡のような最小限の合併症があるはずです。出血が大腿静脈または他の船舶、滅菌綿で適度な圧力の偶発的な破壊によって発生する場合は出血が止まるまでアプリケーターまたはガーゼが出血の部位に適用する必要があるチップを渡した。感染の徴候を示す動物は、抗感染剤で処理されるべきである。重要な壊疽を示す動物はbeeuthanizedする必要がある場合があります。この合併症は、高齢の動物で、そのようなBALB cマウス4など一部の菌株で、より一般的です。さらに、後肢虚血モデルでは、軽度から中程度の痛みが発生することがあります。痛みの治療のために、必要に応じてそのため、このようなブプレノルフィンまたはカルプロフェンのような鎮痛薬がIACUCの勧告によると、投与されるべきである。 結論として、我々は、後肢虚血のマウスモデルにおいてPADを研究するためのシンプルで再現性のある方法を示している。レーザードップラーイメージング解析による後肢虚血モデルでは、血管病理を研究するための、治療の候補を評価するための優れたシステムです。

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murine model of hindlimb ischemia 

米国では、末梢動脈疾患（PAD）は約1,000万人に影響を及ぼし、また普及している世界的です。症状緩和のための薬物療法は限られている。外科手術または血管内介入は、一部の個人のために有用であるが、長期的な結果はしばしば失望させるもの。その結果、PADを治療する新しい治療法を開発するための必要性がある。マウス後肢虚血の準備は、PADのモデルであり、新しい治療法をテストするのに便利です。このような冠動脈や脳動脈の結紮などの組織の虚血の他のモデルと比較すると、大腿動脈の結紮は、虚血組織の単純なモデルのために用意されています。このモデルの他の利点は、大腿動脈と低死亡率へのアクセスが容易です。 このビデオでは、我々は一方的hindimbの虚血のマウスモデルのための方法論を示しています。モデルを作成し、評価するための特定の材料および手順は、レーザードップラーイメージングによる四肢の血流の評価を含め、説明される。このプロトコルは、Huang らによって示されている細胞を、1の移植と非侵襲的な追跡のために利用することができます。

Watch this Scientific Journal Video about Murine Model of Hindlimb Ischemia at JoVE.com

Murine Model of Hindlimb Ischemia

片側後肢虚血の誘発のための外科手術は、レーザードップラー血流イメージングによる虚血の確認を、示されている。

後脚虚血症のマウスモデル

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Research

JoVE Journal

Biology

Murine Model of Hindlimb Ischemia 

57.1K ビュー.  Stanford University. 片側後肢虚血の誘発のための外科手術は、レーザードップラー血流イメージングによる虚血の確認を、示されている。

ビデオ: Murine Model of Hindlimb Ischemia

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

周産期虚血低酸素虚血性脳症モデル

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

生物学

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

後肢虚血の治療のための胚性幹細胞由来血管内皮細胞

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

LAD -ライゲーション：心筋梗塞のマウスモデル

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

マウスにおける伝統的な米航空宇宙局（NASA）後肢アンロードモデルへの交代方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

における虚血/再灌流のための無酸素飢餓モデル<em&gt; C.虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

マウス コリン欠乏エチオニン (CDE) ダイエット慢性肝障害モデル

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

垂直方向のスリーブ胃切除術のマウスモデル

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

同種異系皮膚移植片で再建された火傷のマウスモデル

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

ApoEにおける後肢虚血の改変外科モデル-/- ミニチュア切開を用いたマウス

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...