XHTWは、医学研究でWMケック財団特別若い学者であり、そしてまた、NIH / NHLBIの助成金R01 - HL089598とR01 - R01HL091947、および筋ジストロフィー協会の助成金＃69238でサポートされています。 RJvOは生理ゲノミクスにおける2008年から2010年、アメリカ生理学会ポストドクトラルフェローシップの受賞者です。この作品は、CaMKIIはハートでシグナリングのための財団Leducqアライアンスによって部分的にサポートされています。

パート1：術野の準備<ol><li>動作フィールドは、75％イソプロピルアルコールで消毒されています。 </li><li>加熱パッドは上と右の温度でであることを確認してください。推奨されるシステムは、37℃で維持されている治療のパッドに接続されている水の循環ポンプGaymar℃± 1℃です。心拍数の急激な低下を避けるために手術中に正常な体温を維持することが重要です。 </li><li>手術器具は、手術前にホットビーズ滅菌器で滅菌されています。鈍はさみ、もちろん曲がったピンセットX 2、罰金45 °傾斜鉗子X 2、角度の春のはさみ、胸リトラクタ、およびニードルホルダー：この手順では、以下の手術器具が必要になります。 </li><li>コットンアプリケーターは、出血の場合には手にしてください。 </li></ol>パート2：マウスの​​調製と挿管<ol><li>マウスは0.5 -1.0 L /分100％O 2と混合イソフルラン2％の誘導室内で麻酔する。 </li><li>バリカンは、ネックラインからミッド胸の高さに毛を剃るために使用されます。 </li><li>マウスは、体温を維持するために加熱パッドの上仰臥位に配置されます。 </li><li>ゴムバンドを首を拡張するために動物の前歯の上に置かれる。片手で湾曲した鉗子を使用して、舌を優しく側に操作されます。一方で、気管内挿管はPE 90チューブを使用して実行されます。気管内チューブは、125〜150回/分および0.1〜0.3ミリリットルの一回換気量で、ハーバードのボリュームを再投入齧歯類の人工呼吸器のサイクリングに接続されています。 1.0 L /分100％O 2 -手術中、麻酔は0.5とイソフルラン1.5から2パーセントに維持されている。麻酔の適切なレベルは、マウスの爪床（つま先ピンチ反射）に圧力を適用することによって検証されます。 </li><li>手術野を70％アルコール、続いbetadine液で消毒されています。この手順を3回繰り返されます。 </li><li>運転中に手術野の汚染を防ぐために、滅菌ドレープは、操作フィールドが公開されたまま、マウスの上に配置されています。 </li><li>滅菌手袋のセットは、個々のマウスに使用されます。 </li></ol>パート3：横大動脈のライゲーション<ol><li>第二肋骨までの部分的開胸術は、手術用顕微鏡下で行われ、胸骨は胸のリトラクターを使用して、収納されています。 </li><li>ファイン先端45 °傾斜鉗子を穏やかに大動脈弓から胸腺と脂肪組織を分離するために使用されています。 </li><li>横大動脈の識別に続いて、6.0絹縫合糸の小片は、腕頭と左総頸動脈（図1）の間に配置されます。 </li><li>二つの緩い結び目が横大動脈と27の小片の周囲に関連付けられています½ゲージ鈍針が横大動脈に平行に配置されます。第一結び目はすぐに、針で引き分けた第二に続くと針は速やかに直径0.4mmの収縮を得るために削除されます。偽対照マウスでは、全体の手順は、大動脈のライゲーションを除いて同一です。 </li><li>胸のリトラクタが取り外され、再度膨らませる肺に2秒間人工呼吸器ピンチオフの流出された。 </li><li>胸郭は、結節縫合パターンを6.0 proleneの縫合糸を使用して閉じられます。 </li><li>皮膚は連続縫合パターンを6.0 proleneの縫合糸を使用して閉じられます。 </li></ol>パート4：手術後の回復<ol><li>術後鎮痛のために、マウスは、ブプレノルフィン（0.1 mg / kg）を腹腔内に注入されます。手術後の脱水の兆候がある場合は、滅菌生理食塩水を腹腔内に与えられます。 </li><li>麻酔が徐々にオフの位置に下げ、自発呼吸の兆候が発生したときに気管内チューブは削除されます。 </li><li>マウスは、発生しやすい位置に移動し、加熱パッドを回復するために許可されています。 </li></ol>第5部：横大動脈の成功ライゲーションの確認<ol><li> TAC 1週間後、マウスは横大動脈の結紮により誘発される圧負荷の程度を決定するために再麻酔です。上述したように、麻酔は0.5とイソフルラン1.5から2パーセントに維持されている- 37℃に維持1.0 L /分100％O 2と体温℃± 1℃の</li><li> 20 MHzのドップラープローブは、流速を検出するために45 °の角度で首の左右両側に配置されます。コンピュータベースのドップラー信号プロセッサ（インダスインスツルメンツ、ヒューストン、テキサス州）は、ドップラー信号を表示および格納するために使用されました5。 </li><li>実験的なプロトコルに必要な圧負荷の程度に応じて、一定の範囲内で右頸動脈（RC）/左頸動脈（LC）流量比を持つ唯一のマウスはさらなる分析のために含まれています。深刻な圧負荷を伴う厳密なくびれがtを導くのに対し、例えば、圧負荷中程度は、5-8の比率につながる8-10 OAの比（図5.3A）。シャム動物は（運営が連結していない）、しかし、〜1（図5.3B）の比率が期待される。 </li></ol> 6：代表的な結果野生型マウスでTACを以下の典型的な手術の生存率は約80〜90％です。横大動脈の手術が成功結紮は5-​​10の右と左総頸動脈との間のドップラー流速比（RC / LC）（図1）につながる。偽手術したマウス（図2A）と比較すると、圧負荷を持つマウスは、くびれの気密性（図2B）に応じて、6〜8週後1-2週間以内に心臓肥大、および心拡張を開発することが期待されています。遺伝子組み換えマウスは別の生存率を示すかもしれないがTACの後の最初の月の間に死亡率は、野生型マウスに（&lt;20％）一般的に低いです。6,7 <img src="/files/ftp_upload/1729/1729fig1tmb.jpg" alt="図1" border="0" /> 図1。ドップラー速度計測を用いて横大動脈ライゲーションの確認。右の代表ドップラー速度信号（RC）と左頸動脈（LC）動脈6日後のTAC。これらの録音は、〜6.8のRC / LC流量比で成功したライゲーションを示しています。してください<a href="https://www.jove.com/files/ftp_upload/1729/1729fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <img src="/files/ftp_upload/1729/1729fig2.jpg" alt="図2" /> 図2。 TACに続いて代表的な心臓全体の画像。 。擬似手術野生型マウスの心臓。B。野生型マウスの心臓16週後のTAC。各ライン= 1ミリメートル。

<ol>
	<li>Rockman, H. A., Ross, R. S., Harris, A. N., Knowlton, K. U., Steinhelper, M. E., Field, L. J., Ross, J., Chein, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+atrial-specific+and+inducible+expression+of+an+atrial+natriuretic+factor+transgene+in+an+in+vivo+murine+model+of+cardiac+hypertrophy.">Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.</a> Proc. Natl. Acad. Sci. USA. 88, 8277-8281 (1991).</li><li>Heineke, J., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiac+hypertrophy+by+intracellular+signaling+pathways.">Regulation of cardiac hypertrophy by intracellular signaling pathways.</a> Nat. Rev. Mol. Cell. Biol. 7, 589-600 (2006).</li><li>Hartley, C. J., Reddy, A. K., Madala, S., Michael, L. H., Entman, M. L., Taffett, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+estimation+of+reduced+coronary+flow+reserve+in+mice+with+pressure+overload+cardiac+hypertrophy.">Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy.</a> Ultrasound Med. Biol. 34, 892-901 (2008).</li><li>Reddy, A. K., Taffett, G. E., Li, Y. -. H., Lim, S. -. W., Pham, T. T., Pocius, J. S., Entman, M. L., Michael, L. H., Hartley, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+signal+processing+for+use+in+mice:+applications.">Doppler signal processing for use in mice: applications.</a> IEEE Trans. Biomed. Eng. 52, 1771-1783 (2005).</li><li>Li, Y. -. H., Reddy, A. K., Ochoa, L. N., Pham, T. T., Hartley, C. J., Micheal, L. H., Entman, M. L., Taffett, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+age+on+peripheral+vascular+response+to+transverse+aortic+banding+in+mice.">Effect of age on peripheral vascular response to transverse aortic banding in mice.</a> J. Gerontol. 58A, 895-899 (2003).</li><li>Bourajjaj, M., Armand, A. S., Martins, d. a. C. o. s. t. a., Weijts, P. A., Nagel, B. V. a. n. d. e. r., Heeneman, R., Wehrens, S., Windt, X. H. D. e., J, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NFATc2+is+a+necessary+mediator+of+calcineurin-dependent+cardiac+hypertrophy+and+heart+failure.">NFATc2 is a necessary mediator of calcineurin-dependent cardiac hypertrophy and heart failure.</a> J. Biol. Chem. 28, 22295-22303 (2008).</li><li>Oort, R. J. v. a. n., Respress, J. L., Li, N., Reynolds, C., Almeida, A. C. D. e., Skapura, D. G., Windt, L. J. D. e., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+development+of+pressure+overload+induced+cardiac+hypertrophy+and+dysfunction+in+an+RyR2-R176Q+knockin+mouse+model.">Accelerated development of pressure overload induced cardiac hypertrophy and dysfunction in an RyR2-R176Q knockin mouse model.</a> Hypertension. 55, (2010).</li><li>Tarnavski, O., McMullen, J. R., Schinke, M., Nie, Q., Kong, S., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cardiac+surgery:+comprehensive+techniques+for+the+generation+of+mouse+models+of+human+diseases+and+their+application+for+genomic+studies.">Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies.</a> Physiol Genomics. 16, 349-360 (2004).</li></ol>

人間の大動脈弁狭窄症を模倣TACは、マウスでは心肥大や心不全を誘発することが一般的な方法です。大動脈狭窄のための代替サイトは、昇順および腹部大動脈が含まれています。大動脈狭窄を昇順とは、左心室（LV）上に極端な、より迅速な過負荷を提供します。対照的に、腹部大動脈狭窄は、可能な補償の手段として、循環の無傷の大部分を残します。従って8には、調査へのより?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gaymar T/pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Model TP-500</td>
<td>Circulating water pump</td>
</tr>
<tr>
<td>Temp. therapy pads</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>60-3414</td>
<td>&nbsp;</td>
<tr>
<td>Hot bead sterilizer</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Blunt scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5980</td>
<td>&nbsp;</td>
<tr>
<td>Angled spring scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5668</td>
<td>&nbsp;</td>
<tr>
<td>Chest retractor</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>17002-02</td>
<td>&nbsp;</td>
<tr>
<td>Course curved forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5138</td>
<td>&nbsp;</td>
<tr>
<td>Dumont fine 45&deg; angled forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11253-25</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12565-14</td>
<td>&nbsp;</td>
<tr>
<td>Electric shaver</td>
<td>&nbsp;</td>
<td>General Electric</td>
<td>&nbsp;</td>
<td>For shaving mouse fur</td>
</tr>
<tr>
<td>PE 90 tubing</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427420</td>
<td>For intubation</td>
</tr>
<tr>
<td>MiniVent (ventilator)</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Type 845</td>
<td>&nbsp;</td>
<tr>
<td>Betadine</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19-027132</td>
<td>May be purchased at medical supply store</td>
</tr>
<tr>
<td>Sterile gloves</td>
<td>&nbsp;</td>
<td>Mckesson</td>
<td>20-1565</td>
<td>&nbsp;</td>
<tr>
<td>Stereo microscope</td>
<td>&nbsp;</td>
<td>Unico</td>
<td>ZM186</td>
<td>&nbsp;</td>
<tr>
<td>6-0 silk suture</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18020-60</td>
<td>&nbsp;</td>
<tr>
<td>27 &frac12; gauge needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>6-0 prolene suture</td>
<td>&nbsp;</td>
<td>Mckesson</td>
<td>3286</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Institutional Animal Facility</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transverse aortic constriction in mice

マウスの横大動脈狭窄（TAC）は、圧負荷誘発性心肥大および心不全のための一般的に使用される実験的なモデルです。<sup&gt; 1</sup&gt; TACは、最初は多くの場合、心臓の収縮性の一時的な強化に関連付けられている心臓、の補償肥大につながります。時間が経つにつれて、しかし、慢性的な血行動態の過負荷に対する応答は、心拡張と心不全の結果、不適応となります。<sup&gt; 2</sup&gt;マウスTACのモデルは、最初のロックマンによって検証された<em&gt;ら</em&gt;。<sup&gt; 1</sup&gt;、と以来、広く人間の心血管系疾患を模倣し、心臓肥大応答と心不全の開発に関わる基本的なシグナル伝達プロセスを解明する貴重なツールとして使用されています。このような左前下行（LAD）冠状動脈の完全閉塞などの心不全の他の実験モデル、と比較すると、TACは、心肥大や心不全の発症に、より緩やかな時間経過の再現性の高いモデルを提供します。ここで、我々はマウスに外科TACを実行するためにステップバイステップ手順を説明します。大動脈結紮によって生成圧負荷のレベルを決定するために、高い周波数ドップラープローブは、左右の頸動脈の血流速度との比率を測定するために使用されます。<sup&gt; 3、4</sup&gt; 80〜90％の手術の生存率では、バンディング横大動脈は、マウスの左室肥大や心不全を誘導する有効な手法です。

Watch this Scientific Journal Video about Transverse Aortic Constriction in Mice at JoVE.com

Transverse Aortic Constriction in Mice

マウスの横大動脈狭窄（TAC）は、心肥大や心不全の開発の基礎となるメカニズムを研究するために一般的に使用される実験的なモデルです。ここで、我々は、マウスの心肥大の再現性の学位を作成するために大動脈を収縮する手順を説明します。

マウスの横大動脈狭窄

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Research

JoVE Journal

Biology

66.7K ビュー.  Baylor College of Medicine (BCM). マウスの横大動脈狭窄（TAC）は、心肥大や心不全の開発の基礎となるメカニズムを研究するために一般的に使用される実験的なモデルです。ここで、我々は、マウスの心肥大の再現性の学位を作成するために大動脈を収縮する手順を説明します。

ビデオ: Transverse Aortic Constriction in Mice

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

マウスにおける小腸移植

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

生物学

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

大動脈リングアッセイ

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

マウスにおけるホルター心電図の記録

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

新生児マウスにおける静脈注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

肺動脈収縮による右心室障害の誘導とマウスにおける右心室機能の評価

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

マウスにおけるアピカル歯年理炎の誘導

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

安定化ピン付きマウス大腿骨の横骨折

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

A Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

マウスにおける横行大動脈狭窄の修正技術

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

リン酸カルシウム誘発マウス腹部大動脈瘤モデル

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the ...