This work was supported by grant 13IRG14560023 from the American Heart Association and NIH grants AI078972 and HL108054 to CJO. Support for AOB was from the NIH National Center for Advancing Translational Sciences NIH ULTR001120, and F31 A110417701.

注：すべてのマウス実験は、サンアントニオ（プロトコル＃の13032-34-01C）でのテキサス大学健康科学センターで施設内動物管理使用委員会によって審査され、承認された。動物のケアと公法89から544（動物福祉法）とその改正、公衆衛生サービスガイドライン、および実験動物の管理と使用に関する指針（ヘルス＆ヒューマンサービスの米国務省）に付着した実験プロトコル。 1.感染<ol><li>生後10〜12週齢の間に男女両方のBALB / cマウスを入手します。 注：S. ·ニューモニエ血清型4株をTIGR4は16を配列決定されている毒性の臨床分離株である。 </li><li>光学密度（OD）0.5の620に達するまで、5％CO 2中37℃でトッド-ヒューイットブロス中TIGR4を成長（1.0×10 8コロニー形成単位[CFU] / mlに相当する）。 3で10分間遠心分離することにより肺炎球菌文化をペレット、500 XG及び真空ラインを用いて上清を除去します。サスペンドおよび1.0×10 4 CFU / mlの最終濃度に滅菌リン酸緩衝生理食塩水（PBS）で細菌を希釈する。 </li><li>誘導チャンバーを用いて、酸素中の2.5％イソフルラン気化を有するマウスを麻酔。鈍ピンセットで穏やかな足指のピンチで麻酔状態を確認してください。 <ol><li>その首筋により麻酔マウスを保持し、片手で直立、Sの100μlの腹腔内（IP）を各マウスを注入ニューモニエ懸濁液 （1.0×10 3 CFUのチャレンジ用量に相当する）27〜30ゲージ針で注射器を使用して。バックそのケージにマウスを置きます。マウスは、通常、注射後30〜40秒を目覚めさせる。 </li></ol></li><li>感染が少なくとも24（早期）または30（高度な）時間進行させる。 <ol><li> CO 2窒息によりマウスを安楽死させる。マウスが死亡していることを確認するために頸椎脱臼を行います。安楽死は、さらに除去することによって確認されるステップ1.6で心の。 </li></ol></li><li>アルコール綿棒で尾を消毒2-3 mmのセクションを切り取る。血の2μlのを収集し、連続的に1 U / mlの5回ヘパリンナトリウムを含むPBS中の血液10倍に希釈する。トリプシン大豆血液寒天プレート上にプレート連続希釈し、5％、37℃で一晩インキュベートするコロニーから次の日の外挿2. COは菌血症のレベルをカウントします。 </li><li>外科プラットフォーム上で仰臥位でマウスを固定化。 70％エタノールとパットの乾燥と胸をスプレーしてください。外科用ハサミとピンセットを使用すると、胸腔を開き、胸郭を削除し、心臓や肺を露出するダイアフラムを横断する。ハサミを使用すると、心臓に接続され、血管をカットし、静かにピンセットであざないように注意して心を切り出す。 </li><li> PBSで心を洗い流してから冠状切片が組織切片中に得られるであろうような組織検体採取カセットに入れる。パラフィン包埋、ナンプラーのためにCE 10％緩衝ホルマリン液と次の日におけるカセットは、パラフィン包埋のために送る。 <ol><li>あるいは、ドライアイス上に置かれたクリオモールド内のOCT化合物を用いたフラッシュ凍結。 </li></ol></li></ol>心臓病変および病変内の肺炎球菌の2可視化<ol><li>心臓の4室はそれぞれの組織切片に表示されているようなパラフィン包埋心臓切片を切り倒し。 5μmの厚さでスライドをカット。 </li><li>標準的な方法17を用いて、ヘマトキシリン＆エオシン（H＆E）を用いて脱パラフィン化し、染色スライド。 </li><li>倍率：ビューH＆Eは、低（100X、200X）と高（400X、1,000倍油浸）で光顕微鏡を用いて心臓の部分を染色した。心筋におけるmicrolesionsの存在を心を特徴づける。ここでは、100X 1.3の開口数を計画-NEOFLUARの目標を装備したツァイスAxioskopを2顕微鏡を用いて、H＆E画像を取得する。 注：初期の段階では、心臓のLesionsは、免疫細胞、単球のように見えるもの（単球および顆粒球）の存在下でそれらの差に着色外観によって、場合によっては区別される。進行した病変、30時間で見られる特にでは、免疫細胞が存在せず、大液胞様病変は、その場所で観察されているされている。これは、心臓の病変発生の初期段階での免疫細胞の流入が起こることができるが、それは必要条件ではないように注意することが重要である。 </li></ol> Microlesions内肺炎球菌3.免疫蛍光顕微鏡<ol><li>正に荷電したガラススライド上に5μmのと場所セクションの厚さにミクロトームとcryomoldsのセクション心。凍結された心臓の項では、解凍し、空気乾燥することができます。 10％中性緩衝ホルマリン（25℃でのpHは6.8〜7.2）で10分間のセクションを修正。 </li><li> PBS中0.2％トリトンX-100で15分間透過化、5分間、PBS中のスライド（3×）で洗浄し、次にPBSで再び洗浄する。 </li><li>ブロック組織secti<html> 1時間、PBS中10％ヤギ血清を含むスライド上に。 </li><li> PBSでスライドをすすぎ、カバーと1でウサギ抗血清型4肺炎球菌抗血清を用いて心臓のセクションをインキュベート：PBS中で千37℃で2時間、10％ヤギ血清で。 注：ネガティブコントロールのために研究者らは、ナイーブウサギ抗血清を使用する必要があります</li><li> 37℃で30分間、2000：1で、ヤギ抗ウサギFITCコンジュゲート抗体を、10％ヤギ血清をPBSで切片をPBS、カバーで洗浄し、インキュベートした。 </li><li>製造業者の指示に続いて、真核生物の核の​​可視化のために5mg / mlのDAPI（4 &#39;、6-ジアミジノ-2-フェニルインドール）を使用する。 FluorSaveで組織切片を洗浄し、マウントします。 </li><li>低及び高の両方の倍率で共焦点顕微鏡システムを用いた蛍光画像を取得する。最適な結果を得るために、それに応じて開口数を調整します。ここでは、60X 1.42開口数の対物レンズで共焦点システムを用いた蛍光画像を取得する。 </li></ol>敗血症のマウスの &quot;&gt; 4。抗生物質レスキュー<ol><li>マウスで始まる以前に、ステップ1で示したようにTIGR4の1.0×10 3 CFU を腹腔に感染。 </li><li> 30時間感染後で開始し、100μlの生理食塩水に36時間ごとに12時間を医薬品グレードのアンピシリンIP（80 mg / kg体重）を管理。パラフィン包埋組織、切片のためのステップ1.6で説明したように所望の時点とプロセスでの心を収集します。 </li></ol> 5.コラーゲン沈着の検出<ol><li>キシレンを使用して、標準的な方法17を使用して、脱イオン水、脱パラフィンおよび水和物組織切片をアルコールでの洗浄を段階的。 5分間、水、0.2％リンモリブデン酸とのセクションを治療し、5分間脱イオン水ですすいでください。 </li><li>室温で2時間、ピクリン酸中の0.1％シリウスレッドによる染色切片。 </li><li> 3分間の0.01 N塩酸のセクションを洗浄し、1分間、70％エタノールを用いてすすぐ。 </li><li> Dehydra次いで、キシレンを使用して、同一のセクションをteのが、5.1に概説されたものに洗浄の順序を逆にする、エタノールの濃度を増加させ、段階的に、心臓切片を脱水する。 </li><li>顕微鏡的評価のためのマウントソリューションでマウント組織切片。 </li><li>コラーゲン沈着の領域を同定するために、光学顕微鏡を用いて、低域と高倍率で深い読み取り染色の領域について染色した組織切片を調べる。赤である心臓の心室内の任意の領域は、コラーゲンが堆積された領域である。この赤い染みのない領域はコラーゲン沈着を欠いている健康な組織のサイトです。本明細書において、Picroシリウスレッドを取得するには、20X 0.5開口数の対物レンズを備えた顕微鏡を用いて画像を染色した。 注：健常組織における赤アトリウムの汚れ。 </li></ol>

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The authors declare no conflict of interest in this study.

本報告書では、再現性の高い方法は、Sを誘導する肺炎連鎖球菌は、IPD中に、マウスにおける心臓microlesionsを媒介し、その可視化するための技術が実証されている。マウスは、ヒトの敗血症の間に生じ、心筋内に最終的に細菌のトランスロケーションをもたらすものと同様の高品位菌血症に至るクリアランス免疫系の閾値を超えた細菌の投与量で感染させる。まだ未知の理由のた...

市中肺炎（CAP）のための大人の入院は死亡率1-4に貢献する有害心イベントのために文書化されたリスクを運ぶ。 、心臓の合併症が関連付けられていることが判明、および/ ​​または肺炎に関連した死亡例3の27％を担当した。コラレス·メディナらによる最近の研究では。 肺炎連鎖球菌 （肺炎球菌）、CAPと敗血症5の最も一般的な原因直接入院成人患者6のような多くの19％ほどで有害心イベントと関連していた。肺炎に関連した有害心イベントは、周波数6を減少させるために、新規または悪化うっ血性心不全、不整脈、および心筋梗塞を含む。 Brown らによる最近のPLoSの病原記事で。、肺炎球菌は myocardiuへの参入、心臓の血管内皮を通じて転座が可能であることが判明したmであり、侵襲性肺炎球菌疾患（IPD）7時の心室中の細菌で満たされた離散非化膿性顕微鏡的病変（microlesions）の形成。心臓の微細病変形成の証拠は、肺炎球菌感染症に屈していたヒト以外の霊長類および個人からの心臓の試料で観察された。同様に、実験的に感染させたマウスは、再現性の心臓microlesionsを開発しました。マウスでは、微細病変の大きさと数は積極的に持続し、菌血症、血清中の心筋トロポニンのレベルの重症度、および異常な心臓電気生理学と相関していた。心臓への細菌転移は、血液脳関門および肺炎球菌性髄膜炎の発症、ラミニン受容体および血小板の血管内皮細胞の、すなわち 、コリン結合タンパク質A媒介浸潤横切る肺炎球菌の転座に関与する同じメカニズムを介して起こることが見出された-activating因子受容体依存的に8。 Microlesイオン形成はまた心筋細胞7を殺すことが判明した肺炎球菌孔形成毒素ニューモリシンを必要とした。 肺炎球菌心臓microlesionsは、 黄色ブドウ球菌を含む、他のグラム陽性菌によって引き起こされる化膿性軟部組織および心膿瘍とは区別される。これらは、好中球およびフィブリン沈着9,10により囲まれた細菌の単一の病巣によって特徴付けられる。 S.によって形成されたMicrolesions 肺炎連鎖球菌は 、サイズが小さく、影響を受けた心臓全体に分散、および免疫細胞浸潤を欠いている。感染の初期段階で、microlesionsはS.によって引き起こさ肺炎心筋症の病理学的徴候を彷彿とさせる、損傷または炎症を起こした組織の領域として表示されます。いくつかの単球が、この期間中に観察することができると同時にグラムを継続しながら、しかし、それらの存在は短命であり、病変は急速に外観壊死および細菌で満たさ動物または抗菌介入の死までのサイズでOW。重要なことには、抗生物質の介入の3日以内に、多量の免疫細胞の浸潤は、前者損傷部位で観察され、これは強固なコラーゲン沈着を伴う。同様心臓リモデリングは、心臓機能11-15上の永続的な影響に加えて、以下の梗塞を発生することが報告されています。したがって、microlesionsはIPD中に発生する有害心イベント、おそらく病気のエピソードを生き残った回復期の個体における心臓関連の死亡の発生率の増加のための潜在的な説明である。 ここで、命令は、感染の初期および後期段階でIPD心臓病変形成および心臓microlesionsの可視化の実験的マウスモデルに設けられている。抗菌介入によって保存された動物におけるコラーゲン沈着の検出のためのプロトコルが示されている。この記事の目的は、Fにあるこの重要かつ新規な肺炎球菌病理上の他の研究者の研究をacilitate。

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Todd-Hewitt broth&#160;</td>
			<td style="width:175px;">Neogen</td>
			<td>7161A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:308px;">O.C.T Compound&#160;</td>
			<td style="width:175px;">Tissue-Tek (Sakura Finetek)&#160;</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td style="width:175px;">Fisher Scientific (Acros)</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Normal Goat Serum</td>
			<td style="width:175px;">Abcam</td>
			<td style="width:119px;">ab7481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-serotype 4 pneumococcus antiserum&#160;</td>
			<td>Statens Serum Institut</td>
			<td style="width:119px;">16747</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit FITC conjugated antibody&#160;</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">111-096-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DAPI</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Fluorsave</td>
			<td style="width:175px;">Millipore</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>S70104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ampicillin</td>
			<td style="width:175px;">Sigma</td>
			<td>A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphomolybdic acid 0.2%&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">0.1% Sirius Red in picric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.01 N hydrochloric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Tissue Tack Microscope Slides</td>
			<td style="width:175px;">Polysciences, Inc</td>
			<td style="width:119px;">24216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Paralube Vet Ointment</td>
			<td style="width:175px;">Dechra</td>
			<td>12920060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Hematoxylin and Eosin Staining Kit</td>
			<td style="width:175px;">unspeciified&#160;</td>
			<td style="width:119px;"></td>
			<td>Standard</td>
		</tr>
	</tbody>
</table>

visualization of streptococcus pneumoniae within cardiac microlesions and subsequent cardiac remodeling

菌血症の間、 肺炎球菌は、心筋に血管内皮間で移行して起因する免疫細胞浸潤が存在しない場合に顕著である離散細菌で満たされた顕微鏡的病変（microlesions）を形成することができる。心臓毒性製品の彼らのリリース、Sに起因するmicrolesions内ニューモニエは、しばしば成人における劇症侵襲性肺炎球菌疾患の間に観察される心不全に寄与すると考えられる。本明細書中に30時間以内に心臓の微細病変形成の再現性につながる実験的マウス感染のためのプロトコルを示している。命令はヘマトキシリン​​＆エオシン染色心臓切片で微細病変の同定に提供され、初期および後期microlesions間の形態学的な区別が強調表示されます。命令はSの検証のためのプロトコル上に設けられている肺炎球菌莢膜多糖に対する抗体を用いてmicrolesions内ニューモニエ及び免疫蛍光顕微鏡。最後に、感染したマウスの検出および回復期のマウスの心臓における瘢痕形成を評価するために救助抗生物質の介入のためのプロトコルが提供される。一緒に、これらのプロトコルは、Sへの曝露の結果として肺炎球菌心臓侵略、心筋細胞死、心臓リモデリングの基礎となる分子メカニズムの研究を促進する、肺炎 、心臓における肺炎球菌に対する免疫応答。

H＆EでMicrolesionsの可視化心臓microlesionsはTIGR4との腹腔内チャレンジ以下IPDを有するマウスからの心臓の切片をH＆E染色の心室で観察された。 図1は、ヘマトキシリンより多くて、24及び30時間感染後（hpiの）との間のこれらの病変の大きさの増加を示している病変内に見える-stained（ すなわち 、紫-青）肺炎球菌。 Microlesionsは液胞様の形態、密度の...

Watch this Scientific Journal Video about Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling at JoVE.com

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

の可視化<em&gt;肺炎球菌</em&gt;心臓Microlesionsとその後の心臓リモデリングの中

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

visualization-streptococcus-pneumoniae-within-cardiac-microlesions

Research

JoVE Journal

Medicine

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

8.9K ビュー.  The University of Texas Health Science Center at San Antonio. Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals. 

ビデオ: Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

心臓ストレステストドブタミンによって誘導され、マウスにおける心臓カテーテル検査によって監視される

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

医学

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

直圧モニタリングは正確Cryoballoonアブレーション中に肺静脈閉塞を予測

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

ラットの腹部大動脈の収縮を通じて心臓リモデリングのモデル

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

疑わしい心臓血栓の評価のための心臓磁気共鳴:従来技術と新興技術

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

マウス心ペリサイトの単離と精製

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

無傷心膜虚血性げっ歯類モデル

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Robotized Testing of Camera Positions to Determine Ideal Configuration for Stereo 3D Visualization of Open-Heart Surgery

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room and the surgical field is restricted. It is a sterile environment, and the physical space is crowded with surgical staff and technical equipment. In this setting, unobscured capture and realistic reproduction of the surgical procedures are difficult. This paper presents a method for rapid and reliable data collection of stereoscopic 3D videos at different camera baseline distances and distances of convergence. To collect test data with minimum interference during surgery, with high precision and repeatability, the cameras were attached to each hand of a dual-arm robot. The robot was ceiling-mounted in the operating room. It was programmed to perform a timed sequence of synchronized camera movements stepping through a range of test positions with baseline distance between 50-240 mm at incremental steps of 10 mm, and at two convergence distances of 1100 mm and 1400 mm. Surgery was paused to allow 40 consecutive 5-s video samples. A total of 10 surgical scenarios were recorded.

The human depth perception of 3D stereo videos depends on the camera separation, point of convergence, distance to, and familiarity of the object. This paper presents a robotized method for rapid and reliable test data collection during live open-heart surgery to determine the ideal camera configuration.

開胸手術のステレオ3D可視化のための理想的な構成を決定するためのカメラ位置のロボット化試験

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

心臓評価のための循環死後の献血と正常母性局所灌流の大型動物モデル

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

げっ歯類筋細胞における心収縮機能障害とCa2+ 一過性の解析

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...