NIHのR01 CA128919とNIH R01 HL092923：この作品はエヴァSevickに次の補助金によって支えられている。

すべての動物実験は、それぞれの施設内動物のケアと使用委員会によってテキサス健康科学センター（テキサス州ヒューストン）の大学比較医学科、およびプロトコルの検討および承認後の分子イメージングのためのセンターの基準に準拠して行った（IACUC）または動物福祉委員会（AWC）。 1。イメージングに先立ち24時間動物の作製 リンパイメージングが行われる前に、以下の手順は、日（必要に応じて）実行する必要があります。 <ol><li>インダクションボックスに動物を置き、イソフルランで落ち着いた。 </li><li>動物は深い麻酔（つま先ピンチ操縦で監視）、イソフルランガスに接続されているノーズコーンのおむつ/綿毛パッドと位置鼻の上に場所鎮静動物の状態になると。 </li><li>撮像する領域の周りのすべての髪/毛皮（もしあれば）をクリップします。 </li><li>クリップされた領域への脱毛剤（Nairさん）を適用して、それはO残すnの最大3分までのスキンです。 </li><li>優しく暖かい、湿ったガーゼやペーパータオルですべての脱毛剤を拭き取ってください。 </li><li>優しく暖かい水で皮膚を洗い流し、穏やかにガーゼやペーパータオルを使用して領域を乾燥させてください。 </li><li>動物は加熱パッドの上に置くか、熱ランプの下で回復することができ、それらのケージに戻ります。 </li></ol> 2。イメージングの日<ol><li>その後滅菌水で再構成するの造影剤、cABD-IRDye800ためICGまたは200μM（6.8μg/10μl）のための645μM（5μg/10μl）を達成するために、通常の滅菌（0.85％）、生理食塩水を用いて希釈してください。 暗闇の中で解決策をキープ条件と再構成の6時間内での使用。 </li><li>インダクションボックスに動物を置き、イソフルランで落ち着いた。 </li><li>動物は深い麻酔（つま先ピンチ操縦で監視）、イソフルランガスに接続されているノーズコーンのおむつ/綿毛パッドと位置鼻の上に横向きに場所鎮静動物の状態になると。 </li><li> （その部屋はライトをオフにする暗い）。必要であれば、小さなデスクハロゲンライトは、注射を見るために、少量の光のために使用することができます。 </li><li> 31ゲージの針を有するインスリン注射器を使用して、に応じて、50 ICGのμlまたは各後足および/または尾の付け根の左側と右側の背側面でcABD-IRDye800にID5μlを注入する関心のある領域（説明を参照）。各注射量は0.083〜1.25 mg / kgの（ICG）または0.113から1.7 mg / kgの（cABD-IRDye800）まで及ぶかもしれません。注入量は、動物の系統と注射部位によって異なります。胸腺欠損マウスでは、注入量は5μl（後足）または10μL（尾の付け根）にすることができます。 動物が注射（s）のイメージングシステム下にない場合は、直後の撮像システムの下に動物を置く注射（秒）。 </li><li>全く色素の取り込みがリンパ管に見られない場合は、ステップ2.5は動物プロトコルごとに、必要に応じて繰り返す必要があります。 </li><li>リンパ管が見られたら、黒色の電気テープや黒paで注射部位をカバー当たり。 </li><li>アップV + +ソフトウェアと小動物、NIRFイメージングシステムを用いて1時間にするために、リンパ管の画像を取得する。 （動物はイソフルランで鎮静され、画像が取得されている間呼吸が監視されます。）小動物ながら、NIRFイメージャーは市販されている、我々は、785 nmのレーザーダイオードで構成されるカスタマイズされた、小動物NIRFイメージングシステム（1005〜9ミリメートル-78503を活用、インテンス、ノースブランズウィック、ニュージャージー州）非球面レンズ（C24TME​​-B Thorlabs、ニュートン、ニュージャージー州）、ディフューザー（ED1-C20、Thorlabs）、フィルタ（LD01-785/10-25、Semrock、ニューヨーク州ロチェスター装備）平方センチメートル当たり10未満1.4 mWの入射フルエンス率で動物を照らす一様励振フィールドを作成します。 2 830 nmのフィルター（AND11333、アンドーバー社、セーラム、ニューハンプシャー州）と28ミリメートルニッコールレンズ（1992年、ニコン、との電荷結合素子を電子増倍（EMCCD、PhotonMax512、プリンストンインスツルメンツ、トレントン、ニュージャージー州）のカメラシステムメルヴィル、ニューヨーク州）は、統合ティムとリンパの画像をキャプチャするために使用されスタティックイメージング5のダイナミックイメージングと800ミリ用に200ミリ秒のES。システム構成、各構成要素の詳細については、表、およびキーイメージャの特性の簡潔な議論のための議論については、 図1を参照してください。 </li><li>動物は加熱パッドの上に置くか、熱ランプの下で回復するとそのケージに戻るか、安楽死させることができます。 </li><li> ImageJのか、MATLABソフトウェアを使用して画像を分析します。 図6を参照してください。 </li></ol>

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著者らは、開示することは何もないが、何人かの著者は、特許に記載されています。

我々は、マウスでラベルされたリンパ管の画像をキャプチャするためのカスタム、小動物NIRFイメージングシステムを使用しています。リンパ液の動きのムービーを作成するには、300枚以上の画像が収集されます。映画からリンパ管の機能解析のために、2つ以上のROIは手動でリンパ管に沿って描かれています。 ROIの寸法は、船舶ごとに一定に保たれ、約血管の直径アールアール。動物全体空?...

<table border="1"><tbody><tr><td> ソリューション、試薬、および機器 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>インドシアニングリーン（ICG） </td><td>ペテオンイタリアSpA </td><td> NDC 25431-424-02 </td><td> 645μM（5μg/10μL）に再構成する</td></tr><tr><td>巡回アルブミン結合ドメイン（cABD） </td><td> Bachem社</td><td>カスタム</td><td> 200μM（6.8μg/10μL）に再構成する</td></tr><tr><td> IRDye800 </td><td> LI-COR </td><td> IRDye 800CW </td><td>製造者の指示に従って再構成する。equilmolar濃度でcABDと共役</td></tr><tr><td>滅菌水</td><td>ホスピーラ社、レイクフォレスト、イリノイ州</td><td> NDC 0409-4887-10 </td><td></td></tr><tr><td>ナイル</td><td>チャーチ＆ドワイト社株式会社</td><td>地元の店</td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td>イメージングシステム（以下のコンポーネント） </td><td>分子イメージング研究センター</td><td> N / A </td><td>私たちの研究室で独自に構築しました。 </td></tr><tr><td>電子増倍電荷結合素子（EMCCD）カメラ</td><td>プリンストンインスツルメンツ、トレントン、ニュージャージー州</td><td>光子マックス512 </td><td></td></tr><tr><td>ニコンのカメラレンズ</td><td>ニコン社、メルヴィル、ニューヨーク州</td><td>モデル番号1992、ニッコール28mm </td><td></td></tr><tr><td>光学フィルター</td><td>アンドーバー社、セーラム、ニューハンプシャー</td><td> ANDV11333 </td><td>二つ830.0/10.0 nmのバンドパスフィルタは、レンズの前に使用されている</td></tr><tr><td> 785 nmのレーザダイオード</td><td>激しい株式会社ノースブランズウィック、ニュージャージー州</td><td> 1005-9MM-78503 </td><td>光出力500mWの</td></tr&gt; <tr><td>平行光学系</td><td> Thorlabs、ニュートン、ニュージャージー州</td><td> C240TME-B </td><td>前にクリーンフィルタにコリメートレーザ出力</td></tr><tr><td>クリーンアップフィルタ</td><td> Semrock社は、ニューヨーク州ロチェスター</td><td> LD01-785/10-25 </td><td>蛍光バンドのレーザ発光を削除</td></tr><tr><td>光ディフューザ</td><td> Thorlabs、ニュートン、ニュージャージー州</td><td> ED1-C20 </td><td>動物の上にレーザーを拡散</td></tr><tr><td> V + </td><td>デジタルオプティクス、ブラウンズベイ、オークランド、ニュージーランド</td><td>バージョン5.0 </td><td>ソフトウェアは、カメラシステムを制御し、画像をパソコンに保存するために使用。 <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td>次のソフトウェアパッケージのいずれかを分析ソフトウェアは、画像解析に使用することができます</td></tr><tr><td> ImageJの</td><td>国立Insti健康、ベセスダ、MDのtutes </td><td>入手可能な最新バージョン</td><td>で入手可能なフリーウェア<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> MATLAB </td><td> MathWorks社、ボストン、マサチューセッツ</td><td>バージョン2008年a以降</td><td> <a href="http://www.mathworks.com/" target="_blank">http://www.mathworks.com/</a> </td></tr></tbody></table>

non-invasive optical imaging of the lymphatic vasculature of a mouse

リンパ脈管系は体液の恒常性を維持し、循環器系の重要な構成要素である、腸内の脂肪吸収免疫監視、および仲介を提供しています。しかし、その重要な機能にもかかわらず、リンパ系は、健康と病気の1にこれらの機能を果たすために適応する方法の比較的ほとんど理解があります。最近、我々は、近赤外蛍光（NIRF）染料とカスタムは、Gen III - のトレース管理を使用してリンパ管機能不全を患っている人の中でだけでなく、通常のヒト被験者の行動を &quot;ポンピング&quot;動的イメージリンパアーキテクチャやリンパへの能力を実証しているイメージングシステム2-4を強化した。 NIRFイメージングは​​、ヒトの疾患とリンパのアーキテクチャおよび機能の劇的な変化を示した。それは、これらの変更が発生すると新しい動物モデルは、それらの遺伝的および分子基盤を解明するために開発されているかは不明のままです。このプロトコルでは、NIRFリンパ、sを提示グリーン（ICG）、ヒトの7の50年間使われてきた染料と、優先的にマウスとヒトアルブミン8をバインドNIRF色素標識巡回アルブミン結合ドメイン（cABD-IRDye800）ペプチドを用いたインドシアニンモール動物イメージング5,6 。 ICGに比べて約5.5倍明るく、cABD-IRDye800は類似リンパクリアランスプロファイルを有し、イメージング8のための十分なNIRF信号を達成するためにICGより少ない用量で注射することができる。 cABD-IRDye800と間隙空間8にアルブミンに結合するICG両方あるので、彼らは両方に、リンパ管の中に活性なタンパク質の輸送を示すことができる。皮内（ID）ICGの注射（5-50μL）（645μM）または生理食塩水でcABD-IRDye800（200μM）を各後足および/またはベースの左側と右側の背側面に投与されるイソフルラン麻酔下のマウスの尾。動物で得られた染料濃度は、ICGのため83-1,250μg/ kgの用または113-1,700μg/ kgであるcABD-IRDye800。直ちに注射後、機能的なリンパイメージングは​​、カスタマイズされた、小動物NIRFイメージングシステムを用いて、最大1時間行われる。全体動物の空間分解能は、100ミクロン以下の蛍光リンパ管を描くことができ、深さが3cmに構造の画像アップが9を取得することができる。画像はV + +ソフトウェアを使用して取得すると、ImageJまたはMATLABソフトウェアを使用して分析される。分析時に、全体の血管の直径を包含関心の連続した​​領域（ROI）が与えられたリンパ管に沿って描かれています。各ROIの寸法が与えられた容器とNIRF強度を一定に保たれる定量的血管を通って移動するリンパの &quot;パケット&quot;を評価するために、各ROIを測定する。

マウスにおけるNIRFリンパイメージングの例 ICGまたはcABD-IRDye800が通常のマウスの尾の基部にIDを注入されると、尾の基部に注射部位及び鼠径リンパ節（LN）の間にリンパ脈管はすぐに可視化しなければなりません。まもなく注入後（数秒分）、鼠径LNおよび腋窩LN間のリンパ管は、 図2に示すように可視化されるべきである。彼らは人間で行うようにしたマウスの...

Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

近赤外蛍光（NIRF）を使用して、最近開発されたイメージング技術は、リンパ系の癌転移、免疫応答、創傷修復、および他のリンパ関連疾患に果たす役割の解明に役立つことがあります。

マウスのリンパ脈管構造の非侵襲的光イメージング

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

non-invasive-optical-imaging-of-the-lymphatic-vasculature-of-a-mouse

Research

JoVE Journal

Immunology and Infection

16.0K ビュー.  University of Texas Health Science Center-Houston. 近赤外蛍光（NIRF）を使用して、最近開発されたイメージング技術は、リンパ系の癌転移、免疫応答、創傷修復、および他のリンパ関連疾患に果たす役割の解明に役立つことがあります。

ビデオ: Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

免疫・感染学

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

実験的自己免疫性下垂体炎の誘導のためのマウス下垂体免疫原の調製

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

動的な生細胞イメージングのための宿主 - 病原体相互作用の研究のための光トラップの使用

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

マウス胎児の血管系を標識する方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

マウス膝窩リンパ節の生体内イメージング

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

ゼブラフィッシュ幼生における播種性カンジダ症の非侵襲的イメージング

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

神経病原の非侵襲的モデル<em&gt;エシェリヒア·コリ</em&gt;新生ラットにおける感染

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

非侵襲的気管挿管と成体ラットにおける完全な胸腺摘出

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...