NIH R01 CA128919 ve NIH R01 HL092923: Bu çalışma Eva Sevick için aşağıdaki bağışları ile desteklenmiştir.

Bütün hayvan çalışmaları Texas Sağlık Bilimleri Merkezi (Houston, Teksas), Karşılaştırmalı Tıp Anabilim Dalı, inceleme ve kendi Kurumsal Hayvan Bakım tarafından protokol onayından sonra Moleküler Görüntüleme Merkezi Üniversitesi standartlarına uygun olarak gerçekleştirilen ve Kullanım Kurulu yapıldı (IACUC) veya Hayvan Refahı Komitesi (AWC). 1. Görüntüleme öncesinde 24 Hr Hayvanlar hazırlanması Lenfatik görüntüleme gerçekleşmeden önce aşağıdaki adımları gün (gerektiği gibi) yapılmalıdır. <ol><li> Sıra bir indüksiyon kutusuna hayvan ve izofluran ile oturaklı. </li><li> Hayvan derin anestezi (TEP-tutam manevrası ile izlenmektedir), izofluran gaza bağlı bir burun konisi bir bezi / kabartmak pad ve pozisyon burun üzerinde yer sedasyon hayvan bir devlet olduğunu bir kez. </li><li> Görüntülenecek alanı çevresinde tüm saç / kürk (varsa) klipsleyin. </li><li> Kırpılan alana tüy dökücü ajan (Nair) yağlayın ve o bırakınn 3 dakikaya kadar için cilt. </li><li> Yavaşça sıcak, nemli gazlı bez veya kağıt havlu ile tüm dökücü ajan siliniz. </li><li> Nazikçe ılık su ile cildi yıkayın ve yavaşça gazlı bez veya kağıt havlu ile kurulayın. </li><li> Hayvanlar bir ısıtma yastığı veya bir ısı lambası altında kurtarmak ve kendi kafesine geri dönmek için izin ver. </li></ol> 2. Görüntüleme Günü <ol><li> Steril su ile sulandırın görüntüleme ajanı, ardından cABD-IRDye800 için ICG veya 200 uM (6.8 μg/10 ul) için 645 uM (5 μg/10 ul) elde etmek için steril, normal (% 0,85) salin ile seyreltin. Karanlıkta çözümleri tutun koşulları ve sulandırma 6 saat içinde kullanılmak. </li><li> Sıra bir indüksiyon kutusuna hayvan ve izofluran ile oturaklı. </li><li> Hayvan derin anestezi (TEP-tutam manevrası ile izlenmektedir), izofluran gaza bağlı bir burun konisi bir bezi / kabartmak pad ve pozisyon burun üzerinde yan yeri sedasyon hayvan bir devlet olduğunu bir kez. </li><li> (Yani oda ışıkları kapatınkoyu). Gerekirse, küçük bir masa halojen ışık enjeksiyon görmek için ışık küçük bir miktar için de kullanılabilir. </li><li> Bir 31 gauge iğne ile bir insülin şırıngası kullanılarak bağlı olarak, her bir arka ayak ve / dorsal olarak ya da kuyruk tabanının sol ve sağ tarafında ICG veya cABD-IRDye800 50 ul ID 5 ul enjekte ilgi alanı (Tartışma bakın). Her bir dozu enjekte 0,083 ila 1,25 mg / kg (ICG) ya da 0,113-1,7 mg / kg (cABD-IRDye800) kadar değişebilir. Enjeksiyon hacmi hayvan zorlanma ve enjeksiyon yeri ile değişecektir. Atimik fareler için, enjeksiyon hacmi 5 ul (arka pençe) veya 10 ul (kuyruk baz) olabilir. Hayvan püskürtme (ler) i için görüntüleme sistemi altında değilse, sonra hemen görüntüleme sistemi altında hayvan yerleştirmek enjeksiyon (ler). </li><li> Hiçbir boya alımı lenfatiklerde görülürse hayvan protokol başına gerektiği gibi, adım 2.5 tekrarlanması gerekir. </li><li> Bir kez lenfatikler görülen, siyah elektrik bandı veya siyah pa ile enjeksiyon yerinde kapağıbaşına. </li><li> Kadar kullanarak 1 saat için lenfatik görüntüleri Edinme V + + yazılım ve küçük bir hayvan, NIRF görüntüleme sistemi. (Hayvanlar izofluran ile uyuşturulmuş edilir ve görüntüler elde edilir sırasında solunum izlenir.) Küçük bir hayvan da, NIRF görüntüleyiciler ticari olarak temin edilebilir, bir 785-nm lazer diod oluşan bir özel, küçük hayvan NIRF görüntüleme sistemi (1005 9mm-78503 kullanmak , Yoğun, Kuzey bir asferik lens (C24TME-B, Thorlabs, Newton, NJ), difüzör (ED1-C20, Thorlabs) ile donatılmış Brunswick, NJ) ve filtre (LD01-785/10-25, Semrock, Rochester, NY ) santimetrekare başına 10 daha az 1,4 mW bir olay fluence oranda hayvan aydınlatan bir üniforma uyarım alan oluşturmak için. Bir elektron, iki 830-nm filtreler (AND11333, Andover Corp, Salem, NH) ve 28 mm Nikkor lens (1992, Nikon, kamera sistemi şarj çiftli aygıt (EMCCD, PhotonMax512, Princeton Instruments, Trenton, NJ) çarpılması Melville, NY) entegrasyon tim ile lenfatik görüntü yakalamak için kullanılırstatik görüntüleme 5 için dinamik görüntüleme ve 800 msn için 200 msn es. Sistem yapılandırması, her bileşenin ek ayrıntılar için Tablo ve anahtar görüntüleyici özellikleri kısa bir tartışma için Tartışma için Şekil 1&#39;e bakınız. </li><li> Hayvanlar bir ısıtma yastığı veya bir ısı lambası altında kurtarmak ve kendi kafesine geri dönmek, ya da ötenazi için izin ver. </li><li> ImageJ veya MATLAB yazılımı kullanarak görüntüleri analiz edin. Şekil 6&#39;ya bakınız. </li></ol>

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Yazarlar ifşa etmek başka bir şey var, ama bazı yazarlar bir patent listelenir.

Biz farelerde etiketli lenf damarlarının görüntüleri yakalamak için özel, küçük hayvan NIRF görüntüleme sistemi kullanın. Lenf dolaşımı filmleri oluşturmak için, 300 veya daha fazla görüntü alınır. Filmlerde lenfatiklerin fonksiyonel analizi için, iki veya daha fazla ROI&#39;ler manuel lenf damarı boyunca çizilir. İB&#39;nin boyutları her bir kap için sabit tutulur ve yaklaşık olarak damar çapı vardır. Bütün hayvanlar uzaysal çözünürlüğü 100 mikron veya daha az olan floresan l...

<table border="1"><tbody><tr><td> Çözümler, Reaktifler ve Ekipmanları </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> İndosiyanin green (ICG) </td><td> Patheon Italia SPA </td><td> NDC 25431-424-02 </td><td> 645 uM (5 μg/10 mcL) için sulandırın </td></tr><tr><td> Döngüsel Albumin Bağlama Domain (cABD) </td><td> Bachem </td><td> Görenek </td><td> 200 uM (6.8 μg/10 uL) ile sulandırın </td></tr><tr><td> IRDye800 </td><td> Li-COR </td><td> IRDye 800CW </td><td> Üreticinin talimatlarına göre sulandırın; equilmolar konsantrasyonlarda cABD konjuge </td></tr><tr><td> Steril Su </td><td> Hospira, Inc, Lake Forest, IL </td><td> NDC 0409-4887-10 </td><td></td></tr><tr><td> Nair </td><td> Church &amp; Dwight Co,A.Ş. </td><td> Yerel Mağazaları </td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td> Görüntüleme Sistemi (bileşenleri aşağıda) </td><td> Moleküler Görüntüleme Merkezi </td><td> N / A </td><td> Laboratuvarımızda özel olarak üretilmiştir. </td></tr><tr><td> Elektron-Çarpma şarj bağlı cihaz (EMCCD) kamera </td><td> Princeton Instruments, Trenton, NJ </td><td> Foton Max 512 </td><td></td></tr><tr><td> Nikon fotoğraf makinesi objektifi </td><td> Nikon Inc, Melville, NY </td><td> Model No 1992, Nikkor 28mm </td><td></td></tr><tr><td> Optik filtresi </td><td> Andover Corp, Salem, NH </td><td> ANDV11333 </td><td> İki 830.0/10.0 nm bant geçirici filtre objektif önüne kullanılmıştır </td></tr><tr><td> 785 nm lazer diyot </td><td> Yoğun Ltd, North Brunswick, NJ </td><td> 1005-9MM-78503 </td><td> Optik çıkış 500 mW </td></tr&gt; <tr><td> Collimating optikler </td><td> Thorlabs, Newton, NJ </td><td> C240TME-B </td><td> Öncesinde temizleme filtresi Collimates lazer çıkış </td></tr><tr><td> Temizlik-up filtre </td><td> Semrock, Inc, Rochester, NY </td><td> LD01-785/10-25 </td><td> Floresans bandında lazer emisyonu kaldırır </td></tr><tr><td> Optik difüzör </td><td> Thorlabs, Newton, NJ </td><td> ED1-C20 </td><td> Hayvan üzerinde lazer yayılır </td></tr><tr><td> V + + </td><td> Dijital Optik, Browns Bay, Auckland, Yeni Zelanda </td><td> Sürüm 5.0 </td><td> Yazılım kamera sistemi kontrol etmek ve görüntüleri bilgisayara kaydetmek için kullanılır. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td> Aşağıdaki yazılım paketleri iki analitik yazılım görüntü analizi için kullanılabilmektedir </td></tr><tr><td> ImageJ </td><td> Ulusal EnstitüsününSağlık, Bethesda, MD kitapçığın ikinci bölümüne bakın </td><td> Mevcut en güncel sürümü </td><td> Az SORULAR mevcuttur <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> MATLAB </td><td> MathWorks, Natick, MA </td><td> Sürüm 2008a veya üstü </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

Lenfatik damar sistemi, sıvı dengesinin korur bağırsaktaki immün gözetim ve aracılık yağ emilimini sağlar dolaşım sisteminin önemli bir bileşenidir. Oysa eleştirel fonksiyonuna rağmen, lenfatik sistem sağlığı ve hastalıkları 1 bu işlevlere hizmet adapte nasıl nispeten biraz anlayış vardır. Son zamanlarda, Gen III-, dinamik görüntü lenfatik mimarisi ve lenf gibi iz yakın kızılötesi floresan (NIRF) boya yönetim ve özel kullanarak lenfatik disfonksiyon muzdarip kişilerin yanı sıra normal insan deneklerde &quot;pompalama&quot; işlemi yeteneğini göstermiştir görüntüleme sistemi 2-4 yoğunlaştı. NIRF görüntüleme insan hastalıkları ile lenfatik mimarisi ve işlevi dramatik değişiklikler gösterdi. Bu değişikliklerin ortaya çıkar ve yeni hayvan modelleri kendi genetik ve moleküler temelini gün ışığına geliştirilmektedir nasıl belirsizliğini koruyor. Bu protokol, biz NIRF lenfatik, s sunmakalışveriş hayvan görüntüleme 5,6 green (ICG), insanlarda 7. 50 yıldır kullanılan bir boya ve tercihen fare ve insan albumin 8 bağlayan NIRF boya etiketli siklik albümin bağlanma alanı (cABD-IRDye800) peptid indosiyanin kullanarak . ICG göre yaklaşık 5.5 kat daha parlak, cABD-IRDye800 benzer bir lenfatik açıklık bir profile sahiptir ve görüntüleme 8 için yeterli NIRF sinyal elde etmek için ICG&#39;den daha küçük dozlar halinde enjekte edilebilir. Interstisyel boşluk 8 albümin hem cABD-IRDye800 ve ICG bağlama, her ikisi de içine ve lenfatikler içerisinde aktif bir protein taşıma tasvir edebilir çünkü. İntradermal (ID) ICG enjeksiyonu (5-50 ul) (645 uM) ya da tuzlu su içinde cABD-IRDye800 (200 uM), her bir arka ayak ve / veya tabanının sol ve sağ taraf dorsal ile uygulanırlar Bir izofluran-anestezi fare kuyruğu. Hayvanda çıkan boya konsantrasyonu 83-1,250 ug / kg veya ICG için 113-1,700 mg / kg &#39;dır içincABD-IRDye800. Hemen enjeksiyonları takiben, fonksiyonel lenfatik görüntüleme özelleştirilmiş, küçük hayvan NIRF görüntüleme sistemi kullanılarak en fazla 1 saat için yapılır. Tüm hayvan uzamsal çözünürlük derinliği 3 cm 9 elde edilebilir yapıların floresan lenfatik 100 mikron veya daha az olan gemiler ve görüntüleri tasvir edebilir. Görüntüleri V + + yazılımı kullanılarak kazanılmış ve ImageJ veya MATLAB yazılımı kullanılarak analiz edilmiştir. Analiz sırasında, tüm damar çapı kapsayan faiz (ROI) ardışık bölgeler belirli bir lenf damar boyunca çizilir. Her bir ROI için ölçüler kantitatif lenf damarları yoluyla hareket &quot;paketleri&quot; değerlendirmek için her bir ROI için ölçülür verilen damar ve NIRF yoğunluğu için sabit tutulur.

Farelerde NIRF Lenfatik Görüntüleme Örneği ICG veya cABD-IRDye800 normal bir fare kuyruk dibinde kimlik enjekte, kuyruk dibinde enjeksiyon yeri ve kasık lenf düğümü (LN) arasındaki lenfatik damar hemen görüntülenmiştir olmalıdır. Kısa bir süre sonra, enjeksiyon (birkaç saniye dakika), kasık LN ve koltuk altı LN arasında lenf damarı olarak, Şekil 2&#39;de görüldüğü görselleştirilmiş olmalıdır. Insanlarda olduğu gibi farelerde lenfatik hayvanda...

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

Yakın kızılötesi floresans (NIRF) kullanarak en son geliştirilen görüntüleme teknikleri lenfatik sistem kanseri metastazı oynadığı rol, immün yanıt, yara onarımı ve diğer lenfatik ilişkili hastalıkların ortaya çıkmasına yardımcı olabilir.

Bir Mouse Lenfatik damarları Non-invaziv Optik Görüntüleme

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 Görüntüleme.  University of Texas Health Science Center-Houston. Yakın kızılötesi floresans (NIRF) kullanarak en son geliştirilen görüntüleme teknikleri lenfatik sistem kanseri metastazı oynadığı rol, immün yanıt, yara onarımı ve diğer lenfatik ilişkili hastalıkların ortaya çıkmasına yardımcı olabilir.

Video: 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.

Lökosit Homing Göç ve non-invaziv Görüntüleme In vivo</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 ...

İmmünoloji ve Enfeksiyon

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.

Dinamik Canlı Hücre Görüntüleme Konukçu-Patojen Etkileşimleri Çalışma Optik Tuzak

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.

Embriyonik Farelerde damarları Etiketleme Bir Yöntem

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.

Intravital Görüntüleme Fare Popliteal Lenf Nodu

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.

Zebra balığı Larva yılında Dissemine Kandidiyazis non-invaziv Görüntüleme

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.

Neuropathogenic olmayan İnvaziv Modeli<em&gt; Escherichia coli</em&gt; Yenidoğan Sıçan Enfeksiyon

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.

Non-invaziv Endotrakeal Entübasyon Erişkin Sıçanlarda Komple Timektomi

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.

Bir Zebra balığı Larva Modeli Doğuştan Bağışıklık Tepki Non-invaziv Görüntüleme<em&gt; Streptococcus iniae</em&gt; Enfeksiyon

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.

Farelerde Deneysel Bakteriyel Pnömoni İndüksiyonunun ve fenotipleme bir Non-invaziv ve Teknik Olmayan yoğun Yöntem

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.