Name: intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics
Uploaded: 2015-07-28T13:00:00.000+00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

本研究では、INSERM、エクス·マルセイユ大学、CLフォレスによって得られたHFSPOからキャリア開発賞によってサポートされていました。

倫理文：すべての動物実験は、エクス·マルセイユ大学、フランスの制度動物実験委員会によって承認されたガイドラインおよびプロトコルに従って実施しました。 8での女性のC57BL / 6マウス - 古い10週間は、商業的にDécretN°8 87から848、1987年10月19日、パリの規則に従って取得され、処理されていました。 L.を使用して、すべての実験ドノバン LD1S寄生虫は、フランスと欧州連合の法律の生物学的安全性の規制に従って実施しました。 L. 1.マウスの感染ドノバン前鞭毛寄生虫<ol><li> L.のインビトロで維持するために培地を準備しますドノバン前鞭毛の寄生虫、LD1S（MHOM / SD / 62 / 1S-CL2D）。 <ol><li> M199培地を使用してください。 10％ウシ胎児血清（熱不活性化56℃で30分間）とM199培地サプリメント、25mMのHEPES pHが6.9、12 mMの炭酸水素ナトリウム、1mMのGLUtamine、RPMI 16040ビタミンミックス、10 mMの葉酸、100mMのアデノシン、7.6 mMのヘミン、50 U / mlペニシリンおよび50mg / mlストレプトマイシン。 </li></ol></li><li>文化は、25cm 2の換気フラスコ中で26℃で完全M199培地10ml中の寄生虫をLD1S。 </li><li>連続した差動遠心分離工程を使用して、metacyclic形で濃縮される前鞭毛の寄生虫の集団を準備します。 <ol><li>対数中期寄生虫文化を収穫。 hemocytomerを頼りにしています。完全なM199培地10ml中に5×10 5寄生虫/ mlで再懸濁します。彼らは（約5-6日）で後期定常期に達するまで、嫌気的条件で寄生虫の文化を育てます。 </li><li> 、非metacyclic寄生虫をペレット上清を回収し、10分間26℃で2,500×gで再びそれをスピンする26℃で10分間1200×gでLD1S文化をスピン。 注：この最終ペレットは非常metacyclic前鞭毛の寄生虫に富んでいます。 </li><li> 1にペレットを再懸濁PBSの00μlの。 Vと血球計数器を使用して、寄生虫をカウント：25％グルタルアルデヒドで1/100 Vを固定するための小アリコートを削除します。 </li></ol></li><li> PBS中の1×10 100分の7μlに富む-metacyclic寄生虫集団を希釈します。麻酔したマウスの尾静脈への寄生虫懸濁液を注入30 G針を1mlシリンジを使用して（下記参照）。対照動物では、尾静脈に100μlのPBSを注入。 </li></ol> 2.外科的処置<ol><li> 30分間、70％エタノールで殺生消毒スプレーで作業スペースとすべての手術器具を消毒。 </li><li>ケタミン125mgの/ kgであり、PBSで希釈したキシラジンの12.5ミリグラム/ kgのを含む、動物の体重に応じて、麻酔液を調製します。 </li><li>マウスを計量し、腹腔内に30 G針で1mlシリンジを用いて麻酔薬の適切な投与量を注入します。 </li><li>暖かいマウスを保持し、マウスが完全にanestheであることを確認してください先に進む前に、フットパッドをつまんでtized。マウスでの麻酔液の半分量を再注入するごとに60分。 </li><li>マウスの腹部を剃るとVetedineを使用してクリーニングします。腹部の左側の胸郭下の皮膚や筋肉組織の小さな切開を行います。 </li><li>ちょうど開いた領域の下の腹部に湿ったガーゼを置きます。肝臓を露出させ、ガーゼの上に置きます。肝臓上記湿ったガーゼの別の部分を置きます。 </li><li>顕微鏡下で可視化のために肝臓に、シアノアクリレート結合した金属フレームに、150日24時間、週60ミリメートル厚さ2μmのカバーガラスを配置します。 </li><li>濡れたガーゼでマウスの目を保護します。 </li><li>イメージングセッションの後、頚椎脱臼により麻酔した動物を安楽死させます。 </li></ol>肝臓アーキテクチャの3生体顕微鏡イメージング<ol><li>時を備えた倒立型共焦点顕微鏡で生体顕微鏡の実験を行いますhermostatic室、アポクロマート63Xオイルグリセリン浸対物レンズ（NA 1.4）、アルゴン（488 nm）をヘリウムネオン（543ナノメートル、633 nm）のレーザーと青色ダイオード（405 nm）を制御します。 </li><li>目的に肝臓面を下に覆うカバーガラスと顕微鏡のステージ上にマウスを置きます。 29℃にチャンバーの温度を設定します。正弦波の可視化を可能にするために、肝臓の自己蛍光を使用してフォーカスを調整します。 </li><li>血管系を可視化するために、各マウスについて100μlのPBSで希釈した500μgのBSA-Alexaの647の溶液を調製。 30 G針を有する1mLの注射器を使用して麻酔したマウスの尾静脈に静脈内にこの溶液を注入します。 </li><li>肝細胞核を可視化するために、PBS中のヘキスト33342溶液を調製します。腹腔内に30 G針を1mlシリンジを用いて、マウスに8mg / kgでこの溶液を注入します。 </li><li>肝臓の画像に400 Hzで、通常シーケンシャルモード、63X浸対物レンズとスキャナを使用してください細胞核と肝血管系。ヘキスト励起用の405nmの青色ダイオードとBSA-Alexaの647励起用の633 nmのレーザーをオンにします。アルゴン488 nmレーザーをオンにして、肝臓の自己蛍光を可視化するために大規模な買収帯を定義します。 </li></ol>血流速度測定のための肝臓の4生体顕微鏡イメージング<ol><li>顕微鏡（LAS-AFビューアバージョン3.1.0ビルド8587）のLASソフトウェアを開きます。顕微鏡ソフトウェアを開くときに共鳴スキャナモードを選択します。 </li><li>ハードウェアを設定するには、[設定]タブをクリックします。レーザーをクリックして、ヘリウムネオンレーザ633を選択します。設定をクリックして、12ビットの分解能を選択します。 </li><li>取得メニューをクリックします。ビーム経路の設定]ウィンドウで、目的63Xを選択して、100％に633レーザーパワーを設定します。ドロップダウンメニューPM1-3からのAlexa-633を選択することにより、信号利得をアクティブにし、設定したパラメータをオフにします。 </li><li>取得]タブでは、「XYT」買収を選択モード。 1,024×1024でフォーマット幅を設定します。 8,000 Hzで速度を選択します。 3エアリーユニットのピンホールを選択します。双方向モードを選択しないでください3のズーム倍率を選択します。 </li></ol> RBC速度の5定量分析XYT画像を用いた（方法1） <ol><li>ライブモードでの血液の流れを可視化する「ライブ」アイコンをクリックします。関心のある領域を選択し、分析のための特定の血管を選択します。 USBのコントロールパネルの「X」、「Y」とスキャンフィールド回転座標を調整することにより、水平方向の位置に関心のある血管を設定します。 </li><li>興味のある容器がセットされると、「ライブ」モードを停止します。選択し、ウィンドウ、ライン平均= 1、フレーム平均= 1を設定取得モードで1024×256のフォーマット設定の幅を変更するには、画像を取得するために「スタート」の150をクリックし、「スタック」。 </li><li> 「XYT」画像を使用して、RBC速度の定量分析のためのImageJソフトウェアを開きます。 「ファイル」を選択しますImageJの中のタブには、その後、対象のファイルを選択して「開く」をクリックします。 </li><li> ImageJの中の「プラグイン」メニューに移動し、遺伝子座およびバイオフォーマットオプションをインポート]を選択します。このウィンドウで、「hyperstack」などのパラメータを表示するスタックを選択します。 「xyzct &quot;：スタック順序のオプションでは、オプションを選択します。注：これは、企業買収のすべての一連のウィンドウが開きます。 </li><li> ImageJのメニューの「プラグイン」をクリックし、MTrackJオプションを選択します。小さなウィンドウにある[追加]アイコンをクリックし、追跡するために、RBCをクリックしてください。各次のフレームで同じRBCをクリックして、速度を測定する「尺度」アイコンをクリックしてください。 注：生成されたファイルは.exl形式で保存することができます。 </li><li>容器当たり5 RBCの最小値を追跡し、同じ大きさの3つの個々の血管の最小値を解析します。 </li></ol> XTライン画像を用いたRBC速度の6定量分析（方法2） <ol><li>血液Fを可視化する「ライブ」をクリックしますライブモードで低いです。分析のための特定の血管を選択します。水平位置に関​​心のある血管を設定します。興味のある容器がセットされると、「ライブ」モードを停止します。 </li><li> 「XT」スキャンモードを選択し、ラインスキャンに沿った選択された容器の中央管腔を設定します。 1,024×512で設定された書式の幅、「開始」とXTライン画像を取得の速度= 8,000Hz、ライン平均= 32、時間= 512]をクリックします。 </li><li> .lifファイルを開いて、目的のXTラインの画像を開くことによって、RBC速度の定量分析のための縞を生成します。 LAS-AFソフトウェアでは、「実験」を選択.lifを開いて画像を選択します。注：これは、自動的にカイモグラフを開きます。 </li><li>このカイモグラフ上（X軸上に得られたD、）一定の距離を移動する（ダーク反射を示す）粒子ストリークのために必要な（T、Y軸上で得られる）の時間を測定します。ツール「線を引く」とスジに水平線を描画を選択します。距離に注意してください。μmで、画像の下部に結果タブで生成秒単位の時間。 </li><li>カイモグラフから得られた値を使用して、V =距離/時間として速度を計算します。 </li><li> XTの画像と同じ大きさの3つの個々の容器ごとに最低5粒子筋の最小値を定量化します。 </li></ol>

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The authors have nothing to disclose.

マウス肝臓の生体内顕微鏡検査の最近の開発は、in vivoで 、リアルタイム5,9,10における感染に対する生理的応答の調査のための新しい可能性を開きます。臓器血流量は、しばしば多くの疾患において変化している重大な生理学的パラメータです。しかし、生理学的および感染性条件下での肝血行動態の状態が不十分な探求領域のままです。本研究では、以前に脾臓および腫瘍血...

器官特異的血行動態が、任意の哺乳動物の臓器の重要な生理学的特徴です。血流の異常は、炎症の結果および臓器不全1の徴候である可能性があり。したがって、血流組織、構造および機能は、生理学的および病理学的条件下での分析のためのような重要なパラメータを表示されます。一般的に、特定の器官における血流を解析するために使用されている技術は、技術自体の解像限界を含む、いくつかの制限を含む（ 例えば、血流のドップラーイメージング）当たりの血液のみ（容積絶対血流を測定するための能力臓器を提供するユニット）（ 例えば、光コヒーレンストモグラフィー）および血管2,3の大規模かつ異種集団における速度の平均変化を測定します。肝臓の循環系は、それらのサイズ、構造と機能に不均質である別の容器サブタイプをリンクします。でこの研究は、生体顕微鏡（IVM）イメージング技術は、肝臓器官を構成する個々の血管の特性を明らかにするために、リアルタイムで、高解像度でかつ並列に、インビボでの肝血行動態を評価するために適用されます。この強力な光イメージング技術の最近の開発は、研究者は、高い空間分解能と時間分解能で生きている動物への動的なデータを収集することができます。直接可視化およびインビボでの特異的かつ急速な生物学的過程のリアルタイムの監視を可能にすることにより、IVMは、画像の個々の血管を研究するユニークな機会を提供し、具体的に選択された内の単一の赤血球（RBC）の速度を測定し、定量化します肝臓の血管。 本研究では、肝臓の血行動態上の肝親和性リーシュマニア寄生虫によるマウスの感染の影響を調査するために、マウスの肝臓でのIVMの技術を実装している。L.ドノバンエージェントは、内臓リーシュマニア症、急性·オン·慢性炎症反応と脾臓および肝臓を含む複数の器官で存在する病理学によって特徴付けられる重篤な疾患の原因です。内臓リーシュマニア症の実験マウスモデルでは、脾臓の感染は4プログレッシブであるのに対し、肝臓感染症は、自己解決である。個々の器官に対してリーシュマニア感染のこれらの成果は、まだ完全には理解されていません。病理学的条件の下で、肝臓と脾臓血行動態の検討は、ホスト寄生虫相互作用および疾患の病因に新たな光を当てるします。 我々の実験モデル系は、肝intravasculatureの標識に特異的な蛍光色素の静脈内注射を受け、麻酔したマウスの肝臓を露出し画像化に基づいています。肝臓は、イントラ不可欠な顕微鏡検査のための有利な器官です。小さなincisioを行った後nは腹部に、肝臓を穏やかに外部化され、その後、心拍や呼吸による任意のモーションアーチファクトを低減することを目的としたカバースリップ上に、濡れたガーゼの上に置きました。肝臓は、その後、顕微鏡レンズの視野内に配置されます。 IVMの研究のための2光子顕微鏡を使用する必要が脾臓およびリンパ節と比較して、肝臓の利点は、最大侵入深さで、従来の共焦点顕微鏡の使用を可能にする、その均質な3Dアーキテクチャ/解剖学であります約50μm、生体顕微鏡イメージング5-8。 この研究は、個々の血管におけるRBC速度及び血流速度の定量的測定のための2つの独立した画像化方法を記載しています。第1の方法では、肝血流量は、時間の経過とともに、XY二次元モードを使用して取得されます。得られたXYTデータは個々をの追跡を可能にする無料のImageJソフトウェアでMtrackJプラグインを用いて分析されていますUAL赤血球経時。第2の方法では、単一の血管を選択し、対応する血液の流れは、共焦点レーザー走査顕微鏡のライン走査高速取得モードを使用して分析されます。興味のある容器は、軸線を通って、その中心軸に沿って高頻度で走査されます。血流速度は、次いで、標識されていない暗い赤血球および蛍光標識されたプラズマとの間のコントラストの差に基づいて定量化されます。ラインスキャンに沿って取得された赤血球と血漿の蛍光強度は、角度は、個々のRBCの速度に比例し、筋を得るために時間に対してプロットされています。 この記事の目的は、イメージングのための簡単​​かつ再現性のある方法を提供し、肝臓の個々の血管内の血流速度を測定し、マウスの手術、IVMとの速度の定量分析の成功のパフォーマンスのための基本的なツールを利用できるようにすることです個々の赤血球。 T彼のアプローチは、研究者は、病理学的条件下での血流速度に新たな洞察を得ることができます。

<table><tbody><tr><td>Hoechst 33342</td><td>Sigma Aldrich</td><td>B2261</td><td></td></tr><tr><td>BSA-Alexa 647</td><td>lifetechnologies</td><td>A34785</td><td></td></tr><tr><td>Dextran-FITC 500 mol wt</td><td>SIGMA</td><td>46947</td><td></td></tr><tr><td>Ketamine</td><td>PanPharma</td><td>20434</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>KP07KEU</td><td></td></tr><tr><td>Vetedine</td><td>Pharma Animal</td><td>6869029</td><td></td></tr><tr><td>Cyanoacrylate liquid</td><td>Cyanolit</td><td>5833300005</td><td></td></tr><tr><td>Coverslip frame: Membrane slide for microdissection part N&amp;deg;: 5013</td><td>Molecular machines</td><td>50103</td><td></td></tr><tr><td>Coverslip DiaPath 24x60 ep: 1.6 mm</td><td>DiaPath</td><td>61061</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica&amp;nbsp;</td><td>TCS-SP5</td><td></td></tr><tr><td>LAS-AF viewer&amp;nbsp;</td><td>Leica software</td><td>Version 3.1.0 buid 8587</td><td></td></tr></tbody></table>

intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

生体顕微鏡（IVM）は、リアルタイムで、ライブ動物における様々な生物学的事象の可視化、監視および定量化を可能にした強力な光イメージング技術です。この技術は、非常に生理的プロセスおよび特定の器官中の病原体媒介性の現象の理解を進めています。 本研究では、IVMはマウス肝臓に適用され、プロトコルが設計されている画像に、インビボで肝臓の循環系および個々の肝臓の血管内の赤血球（RBC）の速度を測定します。肝臓器官を特徴付ける別の容器サブタイプを視覚化し、血流速度測定を実行するために、C57BL / 6マウスに、静脈内に、肝臓に関連する血管系を標識する蛍光プラズマ試薬を注射します。 IVMは、in vivo、リアルタイム、関心のある特定の容器内のRBC速度の測定に有効になります。この方法論を確立することが可能になります生理学的および病理学的条件下での肝血行動態を調査します。最終的には、このイメージングベースの方法論は、Lの影響を研究するために重要になります肝血行動態上のドノバン感染。 この方法は、他の感染モデルとマウスの臓器に適用することができ、さらに血流に及ぼす影響を定量化することによって、炎症に対する薬物の効果を試験する臨床を事前に拡張することがあります。

肝臓での正弦波の特定の建築組織が ​​可視化され、この器官（ 図1、パネルBおよびC、緑）、肝細胞核の標識のためのヘキストの腹腔内注射の自己蛍光性に基づくことができる（ 図1B、青）および肝循環系（ 図1C、赤）の染色用蛍光BSAの静脈内注射。肝臓は、直径（D）小血管の（Dで40-700μmの2の範囲の異なる機能や構造や大きさで、いくつかの...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

以下の肝臓の生体顕微鏡イメージング<em&gt;リーシュマニア</em&gt;感染症：肝血行動態の評価

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

intravital-microscopy-imaging-liver-following-leishmania-infection-an

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

11.6K ビュー.  University of Aix-Marseille. This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

ビデオ: Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

免疫・感染学

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

鼠径リンパ節の生体顕微鏡

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

細胞内の抗リーシュマニアに対するスクリーニングのための寄生虫のRescue and形質転換試験<em&gt;ドノバンリーシュマニア</emTHP1ヒト急性単球性白血病細胞株における&gt;無鞭毛

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfpレポーターマウスを使用して、健康で罹患肝臓における免疫細胞の長期生体内多光子顕微鏡イメージング

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

細胞内マクロファージの内部成長の定量化は高速とリーシュ マニア原虫の病原性を評価するための信頼性の高い方法です。

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

マウスの寄生虫毒性を評価するリーシュマニアアマゾネンシスによるVivo感染

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the ...

生物学

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

インビボ ルシフェラーゼを発現するAxenic Amastigotesの歯肉接種による Leishmania乳児感染ハムスターのイメージング

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

以下の肝臓の生体顕微鏡イメージング

要約

さらに動画を探す

この動画の章

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

鼠径リンパ節の生体顕微鏡

脾臓の生体顕微鏡検査：寄生虫モビリティと血流の定量的解析

細胞内の抗リーシュマニアに対するスクリーニングのための寄生虫のRescue and形質転換試験

CXCR6.Gfpレポーターマウスを使用して、健康で罹患肝臓における免疫細胞の長期生体内多光子顕微鏡イメージング

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

細胞内マクロファージの内部成長の定量化は高速とリーシュマニア原虫の病原性を評価するための信頼性の高い方法です。

マウスの寄生虫毒性を評価するリーシュマニアアマゾネンシスによるVivo感染

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

インビボルシフェラーゼを発現するAxenic Amastigotesの歯肉接種による Leishmania乳児感染ハムスターのイメージング

以下の肝臓の生体顕微鏡イメージング

要約

さらに動画を探す

この動画の章

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

鼠径リンパ節の生体顕微鏡

脾臓の生体顕微鏡検査：寄生虫モビリティと血流​​の定量的解析

細胞内の抗リーシュマニアに対するスクリーニングのための寄生虫のRescue and形質転換試験

CXCR6.Gfpレポーターマウスを使用して、健康で罹患肝臓における免疫細胞の長期生体内多光子顕微鏡イメージング

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

細胞内マクロファージの内部成長の定量化は高速とリーシュ マニア原虫の病原性を評価するための信頼性の高い方法です。

マウスの寄生虫毒性を評価するリーシュマニアアマゾネンシスによるVivo感染

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

インビボ ルシフェラーゼを発現するAxenic Amastigotesの歯肉接種による Leishmania乳児感染ハムスターのイメージング

脾臓の生体顕微鏡検査：寄生虫モビリティと血流の定量的解析

細胞内マクロファージの内部成長の定量化は高速とリーシュマニア原虫の病原性を評価するための信頼性の高い方法です。

インビボルシフェラーゼを発現するAxenic Amastigotesの歯肉接種による Leishmania乳児感染ハムスターのイメージング