The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

注：このプロトコルで説明されているすべての動物関連する手順は、以前にデューク大学施設内動物管理使用委員会（DUIACUC）によって承認されている。 1.がん細胞培養およびインジェクション<ol><li>蛍光標識された転移性癌細胞（ 例えば 、ヒトMDAMB231-GFP乳癌細胞、博士パトリシアシュテーク、NCIからの贈り物、とYFP標識マウス肉腫細胞、デビッドキルシュ、デューク大学医療センター、放射線腫瘍科からの贈り物）を育成（10％ウシ胎児血清および1％ペニシリン/ストレプトアビジン、例えばダルベッコ改変イーグル培地（DMEM））適切な培養培地中で、37℃で約90％コンフルエントになるまで。 </li><li>トリプシン処理細胞を、カウント、PBSでそれらを2回洗浄し、次いで27 G針を注射器を用いて、動物あたり5百万個の細胞で、イソフルラン麻酔10週齢の雌ヌードラットの尾静脈へ注入。外科レベルanesthesとりわけ、トーピンチに対する反応の欠如によって確認される。 </li></ol>マイクロCTを使用して、転移の2モニタリング<ol><li>上記転移直径が直径約2mmの存在を検出するために、マイクロCT /マイクロ照射装置を用いて週に一度、ラットを調べる。以前12に記載のように、マイクロCTは、委託されている。 <ol><li>イメージングの前に3％のイソフルラン麻酔の対象とラット。つま先のピンチによる深い麻酔を確認してください。 </li><li>麻酔の発症後、すぐに画像化チャンバ内イメージングクレードルにラットを転送し、2.5から3パーセントにイソフルランと空気の混合物にノーズコーンを経由して接続します。イメージングクレードルの外部に位置制御とレーザー区切り文字を使用して、その胸部がマイクロCTスキャナの光子ビームに配置されている方法で、クレードルにラットの位置を調整します。ガンマ線から調査員を遮蔽するために、イメージング室のドアが閉じていることを確認してください。 </li><li>再び用いて動物の位置を制御するカラービデオカメラ。低解像度のCT撮影テストの実行を実行し、胸腔のXYZ寸法に視野を調整するために、得られる画像を使用しています。 </li><li>イメージラットの40のkVpで2ミリメートルのAlフィルタを使用して、胸郭、2.5ミリアンペア、及び7 fpsで0.008ボクセルとそのケージに動物を返す。 1動物の画像化はしないより15以上または20分を取る必要があります。完全に回復するまで、他の動物の会社に手術を受けた動物を返さないでください。それは胸骨横臥位を維持するのに十分な意識を取り戻したまでは無人の動物を放置しないでください。 </li><li>胸腔内の血管では説明できない、比較的放射線不透過オブジェクトの出現によって転移を確認する（ 図1A） </li></ol></li></ol> 3.ウィンドウ商工手術<ol><li>麻酔、バイタルサイン及び尾静脈カテーテル法<ol><li>転移性疾患の存在で動物を選択します。腹腔dで動物に注入する50mg / kgのペントバルビタールのOSE。先に進む前に、つま先のピンチによって外科レベルの麻酔を確認してください。 注：麻酔プロトコルは、それぞれの実験のセットアップに一致させる必要があります。容易な再​​投薬の選択肢を提供しながらペントバルビタールは、長い手順のための深い麻酔を誘導するために、長時間作用型麻酔薬として、ここで選ばれました。しかし、過剰投与の動物の損失はペントバルビタール麻酔に共通の問題である。ペントバルビタールよりも大きな程度に自律神経反射神経を保持する別のオプションは、そのようなしかし、単一の再投与サイクルのために許可しキシラジンまたはメデトミジン、などの鎮静剤と​​の組み合わせでケタミンです。 </li><li>クリッパーを使用して、転移性疾患を有する、頸部領域における本体の側面に動物を剃る。皮膚から残りのすべての抜け毛を拭き取ってください。抜け毛を除去した後、乾燥するのを防ぐために、眼に獣医軟膏を適用する。 注：無胸腺ヌードラットはrequiが残留髪を持っていること外科手術手順に進む前にRES除去。それは外科的手技およびイメージングを妨げる可能性として、それは、徹底的にすべての毛を除去することは非常に重要です。 </li><li> 37℃の水を循環加熱パッド上に配置された金属板上に仰臥位で動物を修正。フロントと後肢四肢をテープでプレートに固定されている。 注：それは制御し、外科的および実験手順を通じて、パルス酸素濃度計を使用して、このような心拍数と動脈血酸素化などのバイタルサインを記録するのに便利です。 </li></ol></li><li>気管挿管<ol><li>動物の換気のためにカテーテルを配置するために、最初の気管に縦筋系の腹の中央分離することにより横方向子宮頸部皮膚切開を行う。 </li><li>気管の背側を通して縫合糸の通路を作成するには、鋭いピンセットを用いて繰り返し開閉·ツー·クローズアクションを使用します。 </li><li> T上の気管内に小さな切開を作る彼腹側、およそ第二及び第三の気管輪の間に、半円形を超えない。気管カテーテルの固定を可能にするために、背側表面に露出した気管の十分長い部分のままにしておきます。 </li><li>気管内に2.5〜3.0ミリメートル &quot;Y&quot;気管カニューレを挿入し、4-0モノフィラメント縫合糸で締めます。正の肺動脈圧を維持するために、カニューレは水の6センチメートルで満たされている有効期限のダクトに接続され、ボトルに圧力循環人工呼吸器に接続されていることを確認してください。実験的にそれ以外の目的のない限り、流入ガスは、100％酸素でなければなりません。 </li><li> 25-27 G針でカテーテルを挿入し、ラットの尾静脈のいずれかに、ヘパリン化生理食塩水で満たされ、テープで固定します。 注：繰り返し尾静脈にヘパリン処理生理食塩水を少量注入することにより、作業中は尾静脈カテーテルの開存性を確認してください。気管の指導すなわちまた、オロ·気管挿管、麻酔した動物の口を経由し、気管に喉頭過去のチューブは、ここで説明されている気管切開手順に可能な代替である。しかし、この方法は、気管への損傷を避けるために、特別な訓練と経験を必要とし、また、食道の偶発的カニューレ挿入を排除する。 </li></ol></li><li>肺のウィンドウの適用<ol><li>切開を作成することにより、転移性疾患が配置されて胸の側から皮膚を除去した後、鈍的切開を使用して皮膚を外す。 注：皮膚を切除し、剥離の後に除去することができます</li><li>オーバーレイする筋肉組織（胸筋、鋸、および広背筋）の2つの層を切開するが、そのまま肋間筋を残すことによって進んでください。典型的には、2つの隣接するリブの一部を除去することにより、直径約1.5cmの胸腔内の穿孔を作成する。理想的には、第六及び第七rの領域の中に穿孔を見つけるIBS。 </li><li>骨切り術： <ol><li>肺表面に出血し、被害を最小限に抑えるために、しっかりと切削中に歯の手術鉗子で切断するリブを開催。外科用ハサミを使用して、外部のポインティング残存肋骨の尖った側を残して、約45°の角度で、第一リブの内側をカット。 </li><li>その後、再度、肺表面の損傷を防止するために、外側に向いて肋骨の尖った側を残して、同様にして肋骨の側面を切る。 </li><li>肋間筋をカットし、切除した部分を削除した後、隣接するリブのための手順を繰り返します。この手順の間、肺表面と胸郭との間の機械的相互作用が最小化されるように、肺の圧力を維持する。適切に人工呼吸器上のインスピレーションの圧力を調節することによってこれを行います。 </li></ol></li><li>ウィンドウの挿入： <ol><li>あるカバーガラスからなる、カスタムメイドの肺のウィンドウを挿入しますプレキシグラスソケット（ 図1B）に装着。接着によってソケットに窓を取り付け、または真空グリースの非常に少量を適用することによって。表面転移がウィンドウの中心に近い位置していることのようにウィンドウを挿入します。必要に応じて、それぞれの側に少し穴を大きくすることで、ウィンドウの中心に微小転移をもたらすために挿入された穴を調整します。 注：胸膜表面上の転移性疾患は、割れ目に沿って、主に現れる肺表面のそれ以外の場合はピンク·ツー·サーモン色の健全な部品内明確に認識白い点または領域として識別することができます。微小転移は胸膜外装の他の領域で発生する可能性がありますが、調べた細胞株は、ほとんど常に転移性疾患は放射線学的に検出することができたら、穿孔領域に微小転移を浮上表示します。 </li><li>穿孔にソケットに挿入、およびlの臓側胸膜との直接接触を作成した後長官、4.0モノフィラメント縫合糸（ 図1C）を使用して、周囲の肋間筋に、窓枠の縁を縫合する。脱出するとシールを作成するために残留空気を助けるために人工呼吸器にインスピレーション圧力のわずかな増加を使用してください。 注：ラットの呼吸速度は、それは70と90拍の間の呼吸速度を調整することが推奨されるなど、麻酔、興奮や不安状態、吸気中の酸素濃度（のFiO 2）の状態に応じて、大きく変化し得る。インスピレーション圧力が慎重に調整されるべきであり、約以上に設定することはできませ。肺表面への損傷を回避するための8cm H 2 O（0.6 mmHg）で、。 </li></ol></li><li>蛍光顕微鏡下で、恒温（電気）加熱ブランケット上に配置された鋼板上のZ方向の動き（ 図1D）を排除するように設計されているカスタムデザインの制止に動物を配置します。動物の体を制御する直腸サーミスタを用いた温度。動物の制止のネジを調整し、人工呼吸器にインスピレーション圧力が横方向の動きの最適な制御を実現する。 注：二国間、背腹、および頭尾：哺乳動物の自然な呼吸は、肺の動きと胸の延長のすべての3つの方向を伴う。可能な限り自然な呼吸運動を維​​持するためには、必要な範囲でZ方向の圧縮を最小限にすることが重要である。 Z次元拘束血流と他のパラメータに影響を与える可能性のアーチファクトを導入する可能性があるので、同じ動物で繰り返した一連の測定の間、一定の条件を維持することが望ましい。 </li></ol></li></ol>微小循環血流の4イメージングおよび測定<ol><li>心穿刺によって赤血球を収集し、前述のように、のDiI（1,1 = -dioctadecyl -3,3,3- =、3 =テトラメチルインドカルボシアニン過塩素酸塩）でそれらをラベル&lt;SUP&gt; 10。 </li><li>窓室手術が行われる前にガラス窓に初回通過の接着効果を回避するために、ラットの尾静脈への標識赤血球の300μLを注入する。静脈内への空気の導入はそれ以上の注射を阻害するように、注射器やカテーテル内の任意の気泡を除去。 </li><li>画像血液温度を冷却-40℃チップにおける顕微鏡CCDカメラで流れ、及び約100X全体の解像度（10X顕微鏡対物レンズを有する、すなわち 、および10Xプレカメラ眼）。標準ローダミン/ TRITCフィルターセット（励起450から490ナノメートル、放射&gt; 515 nm）を使用してください。結果として生じる画像シーケンスの実際のフレームレートとピクセル解像度を記録する。スタックごとにレコードが少なくとも200（理想的には約300）の画像は、流速の成功分析を確実にする。 </li><li>すべての時間食塩IPの約1ミリリットルを注入することによって、動物における流体損失を補充する。 注：介入を伴う実験的な設定、 例えば肺の癌転移における血流速度を修正する薬剤は、肺転移癌の血流速度の繰り返し測定を必要とする。これらの拡張された実験では、十分な流体を動物に補充することが重要である。 </li><li>尾静脈に3NのKCl 3mlを注入することによって動物を安楽死させる</li><li>すべての血流の流速グレースケール及びカラーコードされたマップ10,11をトレース作成され公開され、公的に利用可能なMatlabのベースのコンピュータアルゴリズムを使用して画像スタックを評価する。続いて、そのようなイメージJとして、商業的または公的に利用可能な画像解析ソフトを用いて得られたグレースケール画像を評価し、血液細胞のない動きがないことを示す値から閾値処理の後、アクティブな脈管構造、すなわちない 。 </li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+theophylline+and+endothelin+receptor+antagonism+improves+exercise+performance+of+rats+under+simulated+high+altitude.">The combination of theophylline and endothelin receptor antagonism improves exercise performance of rats under simulated high altitude.</a> Journal of applied physiology. 113, 1243-1252 (2012).</li></ol>

著者らは、開示することは何もない。

モデルは、生体顕微鏡と計算血流分析を用いて、画像の微小循環血流の変化とラットの肺転移の他の動的プロセスを実行可能であることが示されている。他の方法は、げっ歯類の開放胸郭内の露出肺に顕微鏡検査を実行するために存在するが、このモデルは、閉胸設定における胸壁の穿孔を通して画像肺転移の最初のものである。この方法を使用して、実現可能性は、肺転移の微小循環血流?...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

固形腫瘍における血管系は、屈曲度の大きい度、高い脈管間の距離13を示し、正常な血液供給と大きく異なることが知られている。正常な肺の微小循環（ 図2A、上パネル）と比較した場合、それに応じて、実験的肺乳癌および肉腫の転移における血流トラックは、不規則な形状、大きな脈管間の隙間（ 図2A、下側2パネル）を有する。以前の研究では、肺のウ?...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

ラットの肺転移における微小循環血流速度の自動測定

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K ビュー.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

ビデオ: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

マウス肺内皮表面層の in vivo測定における

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

医学

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

肺気腫とタバコ煙曝露マウスにおける末梢気道リモデリングの自動測定

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

増加肺動脈の拍動流が豚における低酸素性肺高血圧を改善しました

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

早産新生児における経皮微小循環イメージング

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

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 ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

心電図の同時記録と侵襲的血圧ラットにおける測定

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

ラットにおける心筋虚血再灌流後の冠動脈流量の評価

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

ヒトの血流を模倣したフローループモデルにおける血液接触インプラントのヘモ適合性試験

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.