細胞単離後、プロトコル全体で約2時間かかります。 1。顕微準備<ol><li>標準化されたプロトコル9,12に係るドナー骨髄および単離された亜集団の蛍光標識からの幹細胞の単離を行う細胞は、動物の動作が行われる時間の間休止することを許可する。蛍光標識のために我々はCFDA（カルボキシフルオレセインジアセテートスクシンイミジルエステル）をお勧めします。 </li><li>ケタミン（75 mg / kgで）およびキシラジン（2.5 mg / kg）を用いて体重20〜25gの雄マウ​​スを麻酔。外科的手順および実験プロトコール全体を通して、麻酔補助剤によって維持されるべきである（最初の注射の10％、腹腔内）必要に応じて（通常、毎分30〜60）。 </li><li>そっと右陰嚢の前面だけでなく、右太ももと脚の付け根を剃る。すべては湿らせた綿棒で髪を失って収集しています。プレキシガラスの観察ステージ上でマウスの腹側を上に置き、手数料を修正弾性ドレープを使用してT。ステージは、脚と陰嚢の両方を昇降するためのベースプレートの尾側端でベース板と第2板からなる、カスタムメイドされている。 37℃の体温を維持するために加温パッド上にステージを配置ステージ上で固定後、手術用顕微鏡の下で動物を配置し、10を使用して、以下の操作手順を実行します - 16倍の倍率に。 </li><li>鼠径部にまで右太もも、膝から大腿血管のちょうど前方の皮膚のはさみを使って最初の切開を行います。 </li><li>大腿動脈を特定し、周囲の軟組織から動脈を動員し、近位にそれに従ってください。焼灼器を使用して、左睾丸への大規模なブランチを特定し、カット。その後、左睾丸で表面的に滞在縫合を置く。穏やかな牽引力は、大腿動脈の近位のさらなる動員を促進する。 </li><li>近位の動員が完了した後、識別し、大規模なブランチをカット実行中mediall焼灼器による大腿部のY。その後、右大腿部の遠位部での大腿動脈を結紮。合字にやさしい牽引、さらに準備を支援します。 </li><li>再び近位大腿動脈に今の目を向けるなど近位にできるだけ動脈の周りに縫合糸を配置します。結び目を準備しますが、まだそれを接続しないでください。縫合糸に優しいトラクションを適用し、動脈よじれを作成します。 </li><li>よじれを確立した後、動脈が慎重にmicroscissorを使用している切開。大腿静脈を損傷しないように特に注意してください。 </li><li>切開を介して製造マイクロカテーテル（内径0.28ミリメートル）を挿入します。カテーテルは、（0.9％塩化ナトリウム、NaCl）を脱気し、1 mlシリンジ挿入の前に接続されなければならない。 </li><li>カテーテルの挿入が成功した後、慎重にカテーテルの通過を容易にするために、ねじれを緩めます。動脈血流は、カテーテル内ですぐに表示されるはずです。優しくカテーテルを数ミリメートルを進める前のよじれを超えてS。その後、カテーテルの固定のために準備合字を縛り付ける。 </li><li>そっと正しい位置を確認するために、カテーテル（0.9％NaCl）にして吸引をフラッシュします。さらに運用段階にカテーテルを固定するためにテープの一部を使用してください。 </li><li>十分な固定後、精巣挙筋の次の準備ステップのために取ってカテーテルを入れた。血栓形成を防止するため、定期フラッシングとカテーテルごとに5〜10分間の吸引を適用します。 </li><li>右陰嚢に今注目を回して、右陰嚢の腹側面上の皮膚および筋膜を切断することによって切開する。どんな機器とその下の組織に触れないように注意してください。 </li><li>陰嚢の先端に鼠径倍と尾側まで切開を拡張します。 </li><li>それに触れることなく、精巣挙袋上記の基本的なソフトで結合組織を慎重に分離します。精巣挙の先端部を特定し、SLにここに滞在縫合を配置ightly筋肉を伸ばす。優しく尾側と前方の筋肉を引っ張って筋肉の袋の下に今残っている軟組織を切除。 </li><li>結合組織から完全に分離した後、ハ​​サミで頭蓋端に1点で精巣挙袋を開きます。筋肉の先端部に、この開口部から下に切開を延長する。 </li><li>その後、はさみを使って筋肉と精巣の間の残りの接続を切る、焼灼器を使用して睾丸と精巣挙をつなぐ小さな容器を密閉する。 </li><li>顕微鏡分野のうち、睾丸を脇に置きます。 </li><li>開かれた精巣挙は今、ステージの上に平らに産む。精巣挙筋組織を広げるために、組織の端に4 6-0プロレン滞在縫合糸を配置し、弾性ドレープでそれらを修正。これから連続的に1mlシリンジ（ 図1）を用いてリン酸緩衝生理食塩水（PBS）で組織を湿ら。 </li><li>今15分間休む調製を可能にする。その後注入微小血管のコントラスト強調のため左大腿動脈を介して、1％ローダミン-デキストラン0.05〜0.1 mlである。任意のケモカインや炎症剤がアプリケーションのために計画されている場合は、ここで局所的に1ミリリットルの注射器を使用して、それぞれの試薬で筋肉を湿らすべきである。対照実験の場合には、PBSで湿らせ続ける。 </li></ol> 2。生体顕微鏡検査<ol><li>操作ステージに固定されたマウスを維持し、落射照明用に変更し、CCD（電荷結合素子）ビデオカメラに接続され、蛍光顕微鏡下で移動する。大腿動脈カテーテルを脱臼しないよう特に注意してください。 </li><li>水浸対物レンズを用いて実験を行う。 </li><li>精巣挙微小循環の十分な視覚化のために、細胞の注射の前に目標を調整した後、確率的にしっかりした幹細胞の接着の後の分析のために6毛細血管細静脈を定義します。 </li><li>蛍光標識された幹細胞を投与し、大腿動脈カテーテルを利用して5つの連続した注射剤との合計1mlシリンジで、注射当たり0.4×10 6個の細胞で、200μlのPBSに溶解した。慎重に最初の注射前に細胞サンプルを振る。 </li><li>レコードの幹細胞は、それによって典型的なセルラと組み合わせた内皮内層に沿った細胞の速度の50％以上の減少として「ローリング」を定義する、細胞注射の直後にローリング」棒と解放」運動を。ローリング幹細胞の計数およびすべての単一の注射後の観察の1分の間に事前定義された細静脈の距離を通過するすべての幹細胞を実行します。幹細胞ローリングの量は全て通過する細胞の割合として表される。ローリング細胞のようなランダムな簡単なテザリング現象を示す細胞をカウントされません。 </li><li>最後のセルの注射後の計数でしっかりと接着性幹細胞の数を決定し、目の計算された内皮表面に関連して金額を表現接着細胞/ mm 2のようなE 6事前定義された細静脈（直径×長さ×π）。まだ細胞運動が30秒間検出されないときに強固な接着が考えられる。 </li><li>生体顕微鏡が完了した後、慎重に精巣挙筋を取り外し、後で分析（免疫組織化学や分子生物学）に対応して保管してください。頚椎脱臼により動物を生け贄に捧げる。幹細胞のローリングと企業内皮接着、ならびに赤血球速度（100mm /秒）、血管長さ（μm）、血管径（μm）を、壁のシェア率などの微小血管血行動態の定量化のための生体顕微鏡記録の解析（秒-1）;画像処理ソフトを利用し、それぞれの実験組織処置を知らされていない研究者がオフラインで実行する必要があります。 </li></ol>

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この原稿のために行った全ての動物実験は、地元の動物管理使用委員会によって承認されている。 著者は、彼らが競合する経済的利益を持っていないことを宣言します。

<html>生体内蛍光顕微鏡検査は、直接視覚化および幹細胞、内皮相互作用の定量的な分析を可能にする。顕微露光および生体内視覚化技法はよく白血球 - 内皮相互作用および種々の化学成分の研究の間に選択的に単純な表面灌流技術によって標的組織に適用することができる確立されたので、精巣挙筋モデルは、特に有用である。による動きアーチファクトおよび重度画像ノイズが存在しない?...

本資料の目的は、末梢骨髄幹細胞の移動の直接観察および分析のためのマウス精巣挙筋の微小循環に塗布生体顕微鏡（IVM）の技術を記述することである。 幹細胞に基づく組織および臓器再生の現在の概念は、損傷組織1に骨髄由来幹細胞のホーミングを含む。成功した幹細胞の移動のための重要なステップは、経内皮移動、最終的には、臓器移植の2に続いて負傷した臓器内のローカル内皮と細胞の相互作用幹が含まれています。これらの幹細胞の生体内分析-内皮細胞相互作用は、 インビボで異なる病態生理学的な設定での幹細胞に基づく再生応答の定量のために理想的なパラメータを形成する。さらに、それは、考えられる思われる、将来的には、特定の幹細胞pの遊走能の生体内分析標準化された微小環境内opulationsは、さらなる臨床試験をこれらの集団を進める前に適用される可能性があります。 生体顕微鏡は、最初は炎症研究3分野におけるin vivoでの白血球-内皮細胞相互作用を観察し、定量化のために開発されました。生体顕微鏡研究の最初の成功の記録は、光学顕微鏡4の下にカエル」舌や腸間膜を勉強し、19世紀にはすでにコーンハイムから報告された。その最初の使用IVMは、驚異的な技術開発、今日を受けているので、ある種の必須成分は、定量IVMを行うために前提条件を形成は、（i）組織の光アクセスを可能にする製剤、（ii）は、顕微鏡によって検出できる分子プローブ、（三）検出システム、画像データから関心対象のパラメータを抽出することができる（iv）のコンピュータベースの分析システムに接続された顕微鏡4を設定します。 組織標本の様々な腸間膜およびマウスおよびラット5、マウス6、ハムスター、ウサギの耳といくつか例を挙げると、ハムスターの頬袋の背側皮下脂肪チャンバーの肝臓を含むIVM研究のために導入されている。 準備と可視化が十分に標準化された外科的手技によって可能であり、一般的には運動の成果物に問題が発生しないようにただし、次の年には、生体内の観察のための理想的な組織を代表する、マウスの精巣挙筋に焦点を当てます。オープン精巣挙筋標本はバエズや同僚7によって1970年に初めて実施された。当初のラットについて記載し、それはマウス8にも正常に採用されている。これまでの研究は、主に血管壁と白血球の相互作用に焦点を当てた後、私たち自身のグループは最近、評価引当として、マウス精巣挙筋標本を導入直接可視化し、定義微小環境9内の幹細胞内皮相互作用の定量分析のためのBLEツール。様々な幹細胞亜集団は、マウスのc-kit +骨髄幹細胞および間葉系幹細胞、ならびにヒトCD 133 +骨髄幹細胞を10〜12を含む、このモデルを用いて研究されている。ドナー骨髄および可視化のための蛍光標識からの細胞単離後に、幹細胞は、選択的に遠隔の臓器内の任意の細胞の捕捉を回避し、大腿動脈を介して動脈注入を利用して精巣挙筋の微小循環に適用される。潜在的に、それぞれの設定値内の局所幹細胞の遊走を媒介する種々のケモカインは、単純な局所灌流法により標的組織に適用することができるので、精巣挙筋モデルが特に有用である。

<table><tbody><tr><td>Carboxy-fluorescein diacetate succinimidylester (CFDA)</td><td>Invitrogen, Carlsbad, CA, USA</td><td>C1157</td><td></td></tr><tr><td>Rhodamine 6 G (1%)</td><td>Sigma-Aldrich, Munich, Germany</td><td>83697</td><td></td></tr><tr><td>Ketamin (Ketanest)</td><td>Pfizer, Berlin, Germany</td><td>not available</td><td></td></tr><tr><td>Xylacin (Rompun)</td><td>Bayer, Leverkusen, Germany</td><td>not available</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate - buffered saline (PBS)</td><td>PAN Biotech, Aidenbach, Germany</td><td>P04-36500</td><td></td></tr></tbody></table>

intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration

再生細胞治療の文脈における血管細胞のアプリケーションプロトコルの時代において、nonmarrow組織への幹細胞の遊走の基礎となるメカニズムは完全に解明されていない。ここでは、インビボで周辺の骨髄幹細胞の遊走の分析のためのマウスの精巣挙筋の微小循環に適用される生体顕微鏡の技術を記載している。 M.の精巣挙の生体顕微鏡は、以前の血管内皮と白血球の相互作用を直接観察するための炎症研究の分野で導入されている。十分な末梢幹細胞および前駆細胞の移動は、それを裏打ちする内皮に接着沿ってしっかりと圧延同様の初期ステップを含んでいるので内皮とintravasculary投与幹細胞の相互作用の観察および定量化のためM.精巣挙モデルを適用することが考えられる。様々な化学成分を選択的に標的tに適用することができるように初期の幹細胞の動員、骨髄外、生体内の場所を取るための単純な灌流技術による問題は、それが、本質的な微小環境の前提条件を確立することが可能である。

一般的には、精巣挙筋の微小循環内の血管内皮と直接注入幹細胞または前駆細胞の相互作用はまれな事象であると毛細血管細静脈（直径30〜80ミクロン）で独占的に発生します。による蛍光標識圧延しっかりと付着幹細胞に明らかに観察細静脈（ 図2）における内因性白血球の循環から分離して定量化することができる。 血流速度と壁剪断速度で表される微小...

Watch this Scientific Journal Video about Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration at JoVE.com

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

マウスのM.精巣挙微小循環の生体顕微鏡は、ユニークと周辺の骨髄幹細胞の移動を解析するための生体モデルでよく標準化されています。

末梢血幹細胞移動の解析のためのマウス精巣挙筋で微小循環の生体顕微鏡

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

intravital-microscopy-microcirculation-mouse-cremaster-muscle-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.2K ビュー.  University Rostock. マウスのM.精巣挙微小循環の生体顕微鏡は、ユニークと周辺の骨髄幹細胞の移動を解析するための生体モデルでよく標準化されています。 

ビデオ: Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

イントラ不可欠な顕微鏡による骨格筋のin vivo微小循環測定における

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

生物学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

神経科学

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

生体内ビデオ顕微鏡による組織内好中球腔内クロールを追跡、経内皮移動と走

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

免疫・感染学

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

ラットの精巣挙筋の虚血再灌流障害における白血球 - 内皮相互作用のリアルタイムデジタルイメージング（IRI）

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

医学

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

生きたマウスにおける血管炎症及び血栓形成の間に活性化内皮上に異型血小板、好中球の相互作用のリアルタイムイメージング

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

マウスの腸間膜静脈内の生体顕微鏡白血球 - 内皮及び血小板 - 白血球の相互作用

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

リアルタイムで麻酔したマウスに生体顕微鏡により腸間膜静脈に好中球および単球のイメージング

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

微小循環の生体内イメージングのためのマウス精巣挙筋の準備

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

微小循環の生体内蛍光イメージングのためのMicroiontophoresisとマイクロマニピュレーション

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

インビタル顕微鏡で評価されたマウスにおけるクレマスター筋の白血球浸潤

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...