著者らは、機器の胚の開発と利用をスピードアップについてのアドバイスのためにマイクロインジェクショントレーニング、クラリッサ·ヘンリー博士のキャロル·キムの研究室に感謝したい、とFLI1貢献するためのネイサンローソンう：EGFP魚。私たちは、原稿の重要な読書のためにウィーラーラボとショーン壁のメンバーに感謝します。また、このプロジェクトの技術的助言のために魚のケアとアドバイスをマークNilan、とライアンPhennicieとクリスティンガボールに感謝します。この作品は、K.ブラザーズ、MAFESハッチ助成E08913-08、およびR.ウィーラーにNIH NCRR賞P20RR016463にMAFES研究アシスタントシップによって資金を供給された。

利害の衝突が宣言されません。

ここで紹介するゼブラフィッシュマイクロインジェクション法は、Gutzman らとは異なります。ここではその34我々は36から48 HPF幼虫の後脳室に耳胞を介して注入を示しています。我々は記述方法が減少し組織の損傷と後脳の脳室に10月15日酵母の一貫性の注入を可能にします。このプロトコルは、24 HPI（ 図1）と有意な死亡率/罹病率5の結果から、体全?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>産卵水槽</td><td>水生生息地</td><td> 2L </td><td></td></tr><tr><td> 1.7 mLチューブ</td><td> Axygen </td><td> MCT-175-C </td><td></td></tr><tr><td>インスタントオーシャン</td><td>フィッシャー·サイエンティフィック</td><td> S17957C </td><td></td></tr><tr><td>余分深いペトリ皿</td><td>フィッシャー·サイエンティフィック</td><td> 08から757-11Z </td><td></td></tr><tr><td>標準のペトリ皿</td><td> VWRサイエンティフィック</td><td> 25384-302 </td><td></td></tr><tr><td>転送ピペット</td><td>フィッシャー·サイエンティフィック</td><td> 13から711-7M </td><td></td></tr><tr><td>酵母エキス</td><td> VWR ScientiFIC </td><td> 90000-726 </td><td></td></tr><tr><td>ペプトン</td><td> VWRサイエンティフィック</td><td> 90000-264 </td><td></td></tr><tr><td>デキストロース</td><td>フィッシャー·サイエンティフィック</td><td> D16-1 </td><td></td></tr><tr><td>寒天</td><td> VWRサイエンティフィック</td><td> 90000-760 </td><td></td></tr><tr><td>使い捨て血球</td><td> VWRサイエンティフィック</td><td> 82030-468 </td><td></td></tr><tr><td>リン酸緩衝生理食塩水</td><td> VWRサイエンティフィック</td><td> 12001-986 </td><td></td></tr><tr><td>デュモンDumoxelピンセット</td><td> VWRサイエンティフィック</td><td> 100501-806 </td><td></td></tr><tr><td>木製のダボ</td><td> VWRサイエンティフィック</td><td> 10805-018 </td><td></td></tr><tr><td>キムワイプ</td><td> VWRサイエンティフィック</td><td> 300053-964 </td><td></td></tr><tr><td>メルト低いアガロース</td><td> VWRサイエンティフィック</td><td> 12001-722 </td><td></td></tr><tr><td>注射料理のアガロース</td><td> VWRサイエンティフィック</td><td> 12002-102 </td><td></td></tr><tr><td>ブラウンマイクロピペットプラー燃える</td><td>サッター·インスツルメンツ</td><td> P-97 </td><td></td></tr><tr><td>中空ガラス棒</td><td>サッター·インスツルメンツ</td><td> BF120-69から10 </td><td>ブンゼンバーナー上に加熱することにより、滑らかなガラスのガラス棒のために</td></tr><tr><td>ピペットの収納ボックス</td><td>サッター·インスツルメンツ</td><td> BX10 </td><td></td></tr><tr><td> MPPI-3噴射システム</td><td>適用された科学計測</td><td> MPPI-3 </td><td></td></tr><tr><td>背圧ユニット</td><td>適用された科学計測</td><td> BPU </td><td></td></tr><tr><td>マイクロピペットホルダーキット</td><td>アプリエド·サイエンティフィック·インスツルメンテーション</td><td> MPIP </td><td></td></tr><tr><td>フットスイッチ</td><td>適用された科学計測</td><td> FSW </td><td></td></tr><tr><td>マニピュレーター</td><td>適用された科学計測</td><td> MM33 </td><td></td></tr><tr><td>マグネットベース</td><td>適用された科学計測</td><td>マグネットベース</td><td></td></tr><tr><td> Tricaineメタンスルホン酸塩</td><td>西洋ケミカル株式会社</td><td> MS-222 </td><td></td></tr><tr><td>解剖の範囲</td><td>オリンポス</td><td> SZ61トップSZX-ILLB2-100ベース</td><td></td></tr><tr><td>共焦点顕微鏡</td><td>オリンポス</td><td> FV-1000レーザー走査共焦点顕微鏡システムとIX-81 </td><td></td></tr><tr><td> 14インチ試験管ホイールとTC-7組織培養ローラードラム</td><td>ニューブランズウィックサイエンティフィック</td><td> TC-7 </td><td></td></tr><tr><td>イメージング料理</td><td>マテック株式会社</td><td> P24G-1.0-10-F </td><td></td></tr><tr><td>針をロードするためのピペットチップ</td><td>エッペンドルフ</td><td> 930001007 </td><td></td></tr><tr><td>プレート注ぐグリッド</td><td>適応科学ツール</td><td> TU-1 </td><td></td></tr><tr><td>加熱ステージ</td><td> Bioptechs株式会社</td><td>デルタT-5 </td><td></td></tr><tr><td>平らなヘラ</td><td> VWRサイエンティフィック</td><td> 82027-486 </td><td></td></tr><tr><td>プラスチック製のふるい</td><td>ナッツオンラインの用品</td><td> 12センチメートル</td><td></td></tr><tr><td>パラフィルム</td><td> VWRサイエンティフィック</td><td> 52858-000 </td><td></td></tr><tr><td>渦魔神</td><td> VWRサイエンティフィック</td><td> 14216-184 </td><td></td></tr><tr><td> 16×150 mmの培養管</td><td> VWRサイエンティフィック</td><td> 60825-435 </td><td></td></tr><tr><td>光度計</td><td>サーモフィッシャーサイエンティフィック</td><td> ND 2000 </td><td></td></tr><tr><td>フェノールレッド</td><td> VWRサイエンティフィック</td><td> 97062-478 </td><td></td></tr><tr><td>塩酸</td><td> VWRサイエンティフィック</td><td> 87003-216 </td><td></td></tr><tr><td> NaClを</td><td> VWRサイエンティフィック</td><td> BDH4534-500GP </td><td></td></tr><tr><td>塩化カリウム</td><td> VWRサイエンティフィック</td><td> BDH4532-500GP </td><td></td></tr><tr><td> MgSO 4で </td><td> VWRサイエンティフィック</td><td> BDH0246-500GP </td><td></td></tr><tr><td>のCa（NO 3）2 </td><td> VWRサイエンティフィック</td><td> BDH0226-500GP </td><td></td></tr><tr><td> HEPES </td><td> VWRサイエンティフィック</td><td> BDH4520-500GP </td><td></td></tR&gt; <tr><td> Morpholinos </td><td> GeneTools、LLC </td><td></td><td></td></tr></tbody></table>

non-invasive imaging of disseminated candidiasis in zebrafish larvae

病原体カンジダ·アルビカンスに起因する播種性カンジダ症は、入院して個人の臨床的に重要な問題であり、30から40パーセント起因する死亡率は6に関連付けられています。全身性カンジダ症は、通常、先天性免疫によって制御され、食細胞NADPHオキシダーゼなどの自然免疫細胞成分の遺伝的欠陥を持つ個人は、カンジダ7月9日に影響を受けやすくなります。非常に小さなCのダイナミクスについてはほとんど知られていないin vivoでの自然免疫細胞とカンジダの相互作用。in vitro試験で広範には、ホストCの外側にあること確立していますカンジダは、マクロファージの内部で発芽し、かつ迅速に好中球10月14日によって破壊されています。in vitro試験において 、有用なものの、サイトカイン、細胞外マトリックスの添付ファイル、および細胞間の接点10の時間依存ダイナミクスを含む生体内環境で複雑 ​​なを再現することはできません15から18 </sup&gt;。宿主 - 病原体相互作用におけるこれらの要因の寄与を調べるためには、ライブをそのままホストに非侵襲的に感染のこれらの側面を視覚化するためのモデル生物を見つけることが重要です。 ゼブラフィッシュの幼虫は、感染症の研究のためのユニークで多彩な脊椎動物宿主を提供しています。開発ゼブラフィッシュ幼生の最初の30日間だけ先天性免疫防御2、19-21のような先天性免疫に大きく依存している播種性カンジダ症などの疾患の研究を簡素化しています。ゼブラフィッシュ幼生の小型化と透明性の両方の宿主と病原体の細胞レベルでの感染動態のイメージングを可能にします。生得的な免疫細胞を蛍光トランスジェニック幼虫は感染22から24に関与する特定の細胞型を識別するために使用することができます。変更されたアンチセンスオリゴヌクレオチド（Morpholinos）は、そのような食細胞NADPHオキシダーゼなどの様々な免疫成分をノックダウンするとfungaへの応答の変化を勉強するために使用することができますlの感染5。小さな下の脊椎動物を使用しての倫理的、実用的な利点に加えて、ゼブラフィッシュの幼虫は、イメージの両方intravitallyと色の病原体とホストの間で激戦を特有の可能性を提供しています。 ゼブラフィッシュは、ヒトの病原性細菌の数のモデル感染症に使用されており、マイコバクテリア感染3、25の私たちの理解に大きな進歩に尽力してきました。しかし、ごく最近そのような幼虫5に感染するために使用されて菌類、23、26とはるかに大きいの病原体を持っており、感染の方法論の詳細な視覚的な説明がされていない最新の状態に。ここでは、以前のプロトコルへの私たちの修正を含むプリム25ゼブラフィッシュ後脳の脳室のマイクロインジェクションは、当社の技術を紹介します。真菌感染症のために幼虫のゼブラフィッシュモデルを使用して、我々の所見は、in vitro試験から分岐し、宿主-病原体interaを検討する必要性を強化ホストではなく、ペトリ皿5の簡素化、システムの複雑な環境でction。

Watch this Scientific Journal Video about Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae at JoVE.com

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

ゼブラフィッシュの急速な発展、小型サイズと透明性感染症の自然免疫制御の研究のための途方もない利点があります<sup&gt; 1月4日</sup&gt;。ここでは、真菌性病原体を用いたゼブラフィッシュの幼虫を感染させるためのテクニックを実証する<em&gt;カンジダ·アルビカンス</em&gt;マイクロインジェクションすることによって、方法論は、最近、真菌の二形の制御に食細胞NADPHオキシダーゼ活性を巻き込むために使用さ<sup&gt; 5</sup&gt;。

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-imaging-of-disseminated-candidiasis-in-zebrafish-larvae

Research

JoVE Journal

Immunology and Infection

14.6K ビュー.  University of Maine. ゼブラフィッシュの急速な発展、小型サイズと透明性感染症の自然免疫制御の研究のための途方もない利点があります 1月4日。ここでは、真菌性病原体を用いたゼブラフィッシュの幼虫を感染させるためのテクニックを実証するカンジダ·アルビカンスマイクロインジェクションすることによって、方法論は、最近、真菌の二形の?...

ビデオ: Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

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

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

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

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 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 ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

浮袋注入による幼虫ゼブラフィッシュにおける粘膜カンジダ症のモデル化

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

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 ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

非侵襲的<em&gt;インビボ</em&gt;活性化可能な蛍光イメージング剤を用いた炎症性MMP活性の蛍光イメージング

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

結膜から細胞を単離して表現する非侵襲的方法

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...