この研究は、バイオメディカル工学技術センター(BioMET)によって部分的にサポートされました。NIH(1U01HL116321)および(1R01HL142290)および米国心臓協会10SDG4030042(GZ)、19POST34450156(HCJ)。

すべての動物ケアは、メリーランド大学ボルチモア校のガイドラインに従い、制度的動物ケアと使用委員会が承認したプロトコルに従っていました。
1. ソリューションの準備
注: 事前にソリューションを準備してください。実験には、(1)浴過性の生理食塩水(PSS)と(2)ルーメンパーフューズ剤のタイロードの溶液の2種類の基本的なソリューションが使用されています。PSSのpHを維持するためにCO2 による連続バブリングが必要です。HEPES緩衝タイローデの溶液は、気泡が血管に入るのを避けるためにPSSの代わりに内腔で使用され、気泡は内皮細胞7 を損傷し、流れを閉塞させる。
<ol><li>PSS ソリューション: PSS ソリューションの式と構成を表 1に示します。Ca2+フリーでグルコースフリーのPSSストックソリューションを10 Lにし、室温で保管します。1時間は、任意の手順の前に、5%CO2(74%N2および21%O2)で1Lのストック溶液と気泡を取り出し、溶液のpHを〜7.4に保つ。 1.8 g のグルコースと 1.8 mL CaCl2 (1 M ストック)8を追加します。 注:pHが〜7.4のときにバブリングした後にのみCa2+ を追加し、そうでなければ、Ca2+ が沈殿します。</li>
	<li>タイロードの解決策: Tyrodeのソリューションの公式と構成を表 1 に示します。ソリューション(0.22 μm)をフィルターし、4 °Cで保存して、将来の使用のために9.</li>
	<li>タイロードの溶液をBDM(2,3-ブタネジオン単軸)と共に:BDMを可逆筋形筋糸阻害剤として添加して、筋細胞10の収縮を防止する。600 mg の BDM を量り、タイローデの溶液の 200 mL に追加します。.完全に溶解するまで、BDMで溶液をかき混ぜます。それ以上のろ過は必要ありません。</li>
	<li>将来の使用のために4 °CでBDMを含むタイロードの溶液を保管してください。</li>
</ol>2. チャンバーの準備
<ol><li>メーカーの指示に従って、事前に解剖室と実験室の両方をポリジメチルシロキサン(PDMS)でコーティングしてください。</li>
	<li>BDMを含むタイロードの溶液で解剖室を充填します。</li>
	<li>手順の1時間前にチラーをオンにします。温度を4°Cに設定します。</li>
</ol>3. カニューレの準備
<ol><li>マイクロピペットの引き手をオンにします。製造元の指示に従って設定を選択します。</li>
	<li>きれいなホウケイ酸ガラス管を取る(使用したガラス管の外径と内径はそれぞれ1.2mmと0.69mmであった)。</li>
	<li>U字型のプラチナ加熱フィラメントを使用して、サッターピペットプーラーの溝付きクランプにガラスチューブを挿入します。</li>
	<li>引き手をアクティブにして、それぞれに長い薄い先端を持つ2つのカニューレを作ります。</li>
	<li>解剖顕微鏡の下で細かいハサミを使用して各カニューレの先端を切り取り、先端の最終直径を100〜150μm前後にします。</li>
	<li>火はカットカニューレの先端を磨き、鋭いエッジをわずかに丸めます。</li>
	<li>先端から約2mmのシャフトの側面に位置するプラチナワイヤー(細かい制御で加熱)を使用して、シャフトに沿ってカニューレを曲げます。カニューレの曲げ角度は約45°でなければなりません。</li>
	<li>カニューレの開いた端を圧力ミオグラフ室のホルダーに差し込み、フィッティングを締めます。タイロードの溶液を充填した5 mLのシリンジを継ぎ手の反対側に接続します。カニューレをチャンバーに取り付けられたマイクロマニピュレータに接続します( 図1 および 図2を参照)。</li>
	<li>シリンジ(5 mL)を使用してチローデの溶液でカニューレを充填し、それをフラッシュし、空気泡のチューブとコネクタ。</li>
	<li>カニューレ化の手順の前に、所定の流れ範囲にわたってカニューレ内の灌流圧力を測定します(50〜300 μL/min、 図3)。新しいカニューレが使用されている場合にのみ行います。 注:カニューレ内部の灌流圧力は流れに比例し、線形にフィットしています(図3)。</li>
</ol>4. マウス心臓の抽出
<ol><li>イソフルランと酸素のタンクに接続されている麻酔ボックスにC57BL / 6マウス(性別、8-16週齢のいずれか)を入れてください。</li>
	<li>酸素タンクをオンにし、イオフルランの流れを開始します。マウスが完全に麻酔状態になるまで 5 分待ちます。</li>
	<li>麻酔が確立された後、麻酔ボックスからマウスを取り出し、血管構造中の血栓の形成を避けるために、ヘパリンIP(腹腔、360単位、0.5 mL/マウス)を注入します。その後、マウスを麻酔ボックスに戻します。</li>
	<li>ヘパリンを注入してから10分、マウスを上振れしたベッドにスパーンの位置に移動し、ラベリングテープを使用して足を安定させます。鼻コーンを使用してイオブルランで完全な麻酔下にマウスを保ちます。</li>
	<li>マウスの腹部の皮膚を鉗子で横隔膜の上に持ち上げ、手術用はさみを使ってダイヤフラムを切り取り、露出させます。</li>
	<li>横隔膜と胸骨を切ります。胸を開きます。</li>
	<li>胸部腔から心臓を解剖し、胸部壁にできるだけ近くに切断する。</li>
	<li>30 mM BDMを含む氷冷タイロードの溶液に心臓を置きます。</li>
	<li>心臓をきれいにして、肺などの結合組織を取り除きます。</li>
	<li>30 mM BDMを含むタイロードの溶液で満たされた冷やされた解剖チャンバーに心臓を移します。</li>
</ol>5. 中隔動脈の準備とカヌリン
<ol><li>サーボポンプの電源を入れます。サーボポンプを「フロー」モードに設定します。チューブが血管内腔を穿ファミングするために使用されるTyrodeの溶液で満たされるまで、高速で実行してみましょう。チューブに泡がないことを確認します。その後、速度を下げます。</li>
	<li>心臓の中央部分に損傷を与えないように、心臓をPDMSに固定します。</li>
	<li>右と左の両方のアトリアを削除します。右心室を切り開き、解剖顕微鏡を使用して右心室の自由壁を取り除きます。</li>
	<li>中隔動脈を露出し、特定する。ナイロン糸(直径30μm)を中隔動脈の下に置き、将来の使用のために緩い結び目を結びます。 注:中隔動脈は、解剖顕微鏡を使用して容易に識別することができる。中隔動脈は、ほとんどの場合、右冠動脈から起こっている中隔の主要な動脈である。</li>
	<li>細かいはさみを使用して左心室自由壁を取り外します。</li>
	<li>乳頭筋の準備をカニューレが配置された実験室に移す。チャンバーはBDMを含むタイロードの溶液で満たされている。このチャンバの底部は、PDMSの薄い(〜2mm)層でコーティングされています。</li>
	<li>カニューレの位置を調整し(マイクロマニピュレータを使用)、中隔動脈のカニューレを可能にします。結び目を締めます。</li>
	<li>顕微鏡の目的が脈管構造の明確なビューを持っているように部屋の床に小さいピンを使用して乳頭の筋肉を固定します。カニューレの角度と位置に注意を払い、カニューレの先端が動脈壁に平行であることを確認します。</li>
	<li>試験缶取は、徐々にカニューレを介して注射器から溶液を排出することによってカニュール化を行う。すべての要素が正常に機能する場合, 乳頭筋の残留血液は、このプロセスの開始時に組織を出て、唯一のタイローデの溶液は後で見られます.</li>
</ol>6. 準備の安定化
<ol><li>過灌流液を提供する蠕動ポンプをオンにします。その後、常に3-4 mL /分の速度でプレガスPSSでバス内の準備を過熟させる。</li>
	<li>スーパーフュージョン溶液の温度コントローラをオンにします。浴の温度を~35~37 °Cに調整してください。</li>
	<li>カニューレをサーボポンプに接続します。圧力サーボコントローラに表示される圧力を読み取ります。</li>
	<li>流れと圧力を監視します。流量(μL/分)と動脈灌流圧(mmHg)はデジタイザを使用してデジタル化され、関連するソフトウェアを使用して記録される(図2)。</li>
	<li>流れを調整して圧力を10mmHgに設定し、約10分間動かします。</li>
	<li>明るい溶液の流れを増やして、動脈の圧力を60mmHgにします。カニューレの圧力降下を差し引くことによって、動脈自体の最終的な灌流圧を得る(図3)。 メモ:圧力サーボコントローラの「圧力」モードを選択した場合、これらの実験では、発光圧力が〜60mmHgに設定されます。次に、フローの変化を監視します。</li>
	<li>流れや圧力レベルを監視して、サンプルを30~60分間安定させます。動脈圧の漸進的な増加は、通常、カヌリン化後の灌流の開始時に観察される。新しい定常状態は、約30分でそれ自体で確立されます(図4)。</li>
	<li>灌流圧力が安定した後(一定の流れで)実験を開始します。</li>
</ol>7. 蛍光タグ付き小麦胚芽凝集剤(WGA)で調製物をロードする
<ol><li>アレクサ-フルーオール-488を含むタイロードのソリューションを調製し、100 μgの共役WGAをタイロードの溶液の5 mLに加えます。</li>
	<li>通常のタイローデの溶液から蛍光タグ付きWGAを含む溶液に慎重にパーフューズを切り替えます。気泡が導入されないように、慎重にパーフューザートを切り替える必要があります。</li>
	<li>WGA灌流の〜30分後、またはWGA溶液がなくなったとき、パーフューズを通常のタイロードの溶液に戻します。</li>
</ol>8. 細動脈と毛細血管の共焦点イメージング
<ol><li>ニポウディスク共焦点顕微鏡システムをオンにします。</li>
	<li>顕微鏡を使用して、透過光モードで低電力目標(4x)を使用して中隔動脈を見つけます。 注:中隔動脈はカニューレの位置に従うことによって見つけることができる。</li>
	<li>回転ディスク共焦点で共焦点イメージングを開始し、488 nm励起レーザー、40倍の客観的な倍率を選択し、レーザー強度とサンプリングレート(画像あたり10〜500 ms)を調整します。</li>
	<li>共焦点顕微鏡の上と下のイメージング位置を設定して、イメージング範囲を定義し、zスタックイメージングのパラメータを入力します。</li>
	<li>使用しているシステムに推奨されるステップサイズを設定するか、ステップサイズを必要に応じて設定します。</li>
	<li>Z スタック設定または時系列設定を選択します。</li>
	<li>新しいイメージを保存する新しいフォルダを選択または設定します。</li>
	<li>[実行] をクリックして、後で分析するためにイメージングの記録を開始します (図 5)。</li>
</ol>9. 血管拡張薬実験例:ピンアシチル誘発血管拡張(ビデオ1)。
<ol><li>ピンアシチルストック溶液(DMSOで100 mM)を準備し、4°Cで保存します。</li>
	<li>タイロードの溶液の10 mLを準備します。ピンアシチルストック溶液を10 μL加え、ピンアシチルの最終濃度を100 μMにします。</li>
	<li>画像は、低倍率(4x目的)で乳頭筋を中隔動脈および枝動脈を含むようにする。</li>
	<li>発光溶液をピンアシチル含有溶液に切り替える。</li>
</ol>10. 血流制御実験例:血管収縮剤誘発動脈灌流圧が一定の流れで増加する(図6)
<ol><li>エンドセリン-1(ET-1)ストック溶液(10 μM)を調製し、-20°Cで保存します。</li>
	<li>タイロードの溶液の10 mLを準備します。ET-1ストック溶液(10μM)を10 μL加え、ET-1 10 nMの最終濃度を実現します。</li>
	<li>一定の流れで発光圧を記録し、乳頭筋を画像化します。</li>
	<li>発光溶液をET-1含有溶液に切り替える。</li>
</ol>11. 毛細血管とペリサイトの画像例 (図7)
<ol><li>このプロトコルのセクション4に記載されているようにNG2DsRedBACトランスジェニックマウスを犠牲にします。</li>
	<li>心臓を抽出し、中隔動脈をカニューレ化し、Alexa-Fluor-488コンジュゲートWGAを5-7プロトコルでサンプルに負荷をかける。</li>
	<li>画像488nmおよび560 nm励起で乳頭筋、および510-525 nm、575-625 nm発光を共焦点を用いた。動脈圧は約40mmHgになります。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+in+vitro+approach+to+study+neurovascular+coupling+mechanisms+in+the+brain+microcirculation.">Advanced in vitro approach to study neurovascular coupling mechanisms in the brain microcirculation.</a> Journal of Physiology. 590, 1757-1770 (2012).</li></ol>

本研究では、生理的条件下で心臓の冠状微小循環を研究する非常に単純でありながら非常に実用的なex vivo法を導入しました。この方法は、ラット2を用いた機械的調査から改変した。その他、高速かつ高い光学分解能を備えたイメージング技術が実現しました。そのため、現在市販されている高度な光学イメージングシステムを利用することができました。機能する乳頭筋?...

適切な冠状動脈圧流制御は、その代謝要求を満たすために心臓に十分な血液供給を保証する 1.しかし、過去数十年間に生体内およびインビトロで行われてきた広範な研究にもかかわらず、冠状動脈圧流が心臓でどのように動的に調節されているかは、最近になってようやく明らかになりました。その理由の一つは、心臓の絶え間ない鼓動によるこのような研究のための生理学的作業モデルの確立の難しさである。いずれにせよ、生体組織や動物における冠状動脈微小血管の観察には種々の方法が確立されているが、これらの方法のいずれも、圧力、流れ及び微小血管径の測定と一定/安定的な焦点を達成できなかった2,3。心臓を鼓動する冠動脈微小血管の直接可視化は数十年前に導入されました4,3, 小血管の直径の測定は困難であり、微小循環に関連する多くの特殊な細胞タイプの特定の機能は同様に厄介でした.ストロボスコピック法と浮遊目的システムでさえ、上記の情報を同時に提供できませんでした5.それにもかかわらず、前述の技術を用いてかなりの量の貴重な情報が得られ、冠状血流の調節に関する理解を深めるのに役立っています。この論文で説明する方法は、冠状動脈、細動脈および微小血管系の成分が刺激および代謝要求に対してどのように反応するかを調査し、詳細に理解するのに役立つ。
これらの研究を追求するために確立した作業モデルは、前作のWesterhofらの2に基づいて構築された。マウス心臓の中隔動脈のカヌル化後、生理食塩水は、筋細胞および心臓組織の他の成分を栄養を保つためにその動脈を浸透させるために使用された。動脈灌流圧、流れ及び血管径は、適切な蛍光指標を用いて他の生理学的機能の中でモニターした。この方法により、生体組織の生理的圧力下で冠状動脈性微小血管床を可視化し、初めて微小循環調節の基盤となる細胞機構を研究することが可能になります。

<table>
	<tbody>
		<tr>
			<td>1 M CaCl2 solution</td>
			<td>MilliporeSigma, USA</td>
			<td>21115</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2 solution</td>
			<td>MilliporeSigma, USA</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>AxoScope software</td>
			<td>Molecular Devices, San Jose, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chiller/water incubator</td>
			<td>FisherScientific, USA</td>
			<td>Isotemp 3016S</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal</td>
			<td>Nikon Instruments, USA</td>
			<td>A1R</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom glass tubing</td>
			<td>Drummond Scientific Company</td>
			<td>9-000-3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1322A</td>
			<td>Molecular Devices, San Jose, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus, Japan</td>
			<td>SZX12</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>MilliporeSigma, USA</td>
			<td>E7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt</td>
			<td>Sigma-Aldrich, USA</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline solution Heater</td>
			<td>Warner Istruments, Hamden, CT, USA</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETone, Idaho, USA</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments, Novato, CA, USA</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette/cannula holder</td>
			<td>Warner Istruments, Hamden, CT, USA</td>
			<td>64-0981</td>
			<td></td>
		</tr>
		<tr>
			<td>NG2DsRedBAC transgenic mouse</td>
			<td>The Jackson Laboratory</td>
			<td>#008241</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon thread for tying blood vessels</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>THR-G</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS (polydimethylsiloxane)</td>
			<td>SYLGARD, Germantown, WI, USA</td>
			<td>184 SIL ELAST KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>minipuls 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Servo Controller</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>PS-200-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Scientific Tools, Foster City, CA, USA</td>
			<td>15000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Servo Pump</td>
			<td>Living Systems Instrumentation, Burlington, Vt, USA</td>
			<td>PS-200-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td>
			<td>ThermoFisher Scientific, Waltham, MA USA</td>
			<td>W11261</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic measurement and imaging of capillaries, arterioles, and pericytes in mouse heart

毛細血管の開閉と共に冠状動脈の緊張は、一定の灌流圧で心筋細胞への血流を主に決定する。しかし、心臓全体の冠状動脈と毛細血管の動的変化を監視することは困難である, 主にその動きとノンストップの鼓動.ここでは、マウス右心室の乳頭筋における動脈灌流速度、圧力、および動脈および毛細血管の直径変化をモニタリングできる方法について述べている。マウス中隔動脈は、一定の流れまたは圧力でカニューレ化され、他の動的に測定される。蛍光標識レクチン(例えば、アレクサ・フルオール-488または-633標識ウィート・ゲルム・グルチニン、WGA)で灌流した後、右心室の乳頭筋および中隔の動脈および毛細血管(および他の血管)を容易に画像化することができた。血管径の変化は、心臓収縮の有無において測定することができる。遺伝子組み換え蛍光タンパク質が発現した場合、特定の特徴を監視することができた。例えば、NG2-DsRedを発現したマウス心臓でペリサイトを可視化した。この方法は、心臓の毛細血管のペリサイトの生理機能を研究するための有用なプラットフォームを提供してきた。血管/毛細管径と動脈の光圧を同時に測定することで、心臓の血流に対する試薬の効果を研究するのにも適しています。この調製物は、最先端の光学画像システムと組み合わせることで、ほぼ生理学的条件下で心臓の細胞および分子レベルで血流とその制御を研究することを可能にする。

蛍光血管マーカーが血管内腔に浸透する場合(ここでWGAはAlexa Fluor-488と共役)、高速共焦点顕微鏡を用いて 図5( 左パネル)に示すように、全血管樹木を可視化することが可能です。さらに拡大すると、毛細管の撮像を詳細に行うことができます(図5、右パネル)。加圧システムは、光圧の一定のモニタリングをサポートするので、この調製物は動脈圧と...

Watch this Scientific Journal Video about Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart at JoVE.com

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

ここでは、中隔動脈がカニュール化および加圧されるように、動脈灌流圧および流れを維持する動脈灌流圧および流れのex vivoモニタリング、ならびに毛細血管床および周囲細胞を含む血管樹木成分のエキビボモニタリングによって、生きているマウス心臓組織における冠状微小循環を研究するためのプロトコルである。

マウス心臓における毛細血管、細動脈、および周囲細胞の動的測定とイメージング

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

dynamic-measurement-imaging-capillaries-arterioles-pericytes-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

5.3K ビュー.  University of Maryland School of Medicine. ここでは、中隔動脈がカニュール化および加圧されるように、動脈灌流圧および流れを維持する動脈灌流圧および流れのex vivoモニタリング、ならびに毛細血管床および周囲細胞を含む血管樹木成分のエキビボモニタリングによって、生きているマウス心臓組織における冠状微小循環を研究するためのプロトコルである。

ビデオ: Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

定量的な可視化と動的な熱画像を用いた皮膚癌の発見

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

医学

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

X線CTを用いたマウス肥満モデルにおけるセグメンテーションおよびFATボリュームの測定

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

乳幼児の脳代謝と血行動態の非侵襲的光計測

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

非侵襲Mモード心エコー検査によるマウスでの心拍数の子宮内測定で

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

マウス絞り機能の診断用超音波イメージング

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

マウス網膜細動脈Ex Vivoでの反応性の測定の準備手順

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

顔面性腰痛患者における動的骨回転率の評価に関する定量的[18F]-Naf-PET-MRI分析

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

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 ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

マウスの洞房結節と房室結節のホールマウント免疫蛍光染色、共焦点イメージング、3D再構成

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...