Bu çalışma NIH hibe R01-3.155-53.502 ve Washington Üniversitesi-Pfizer Biyomedikal Araştırma Anlaşması tarafından desteklenmiştir.

1. Hayvan hazırlanması: Bu protokol, nötrofil ve makrofajların floresan eGFP 1 ifadesi etiketli olan yetişkin dişi yetişkin LYSM eGFP fareler kullanır. <ol class="lalpha"><li> Kan damarlarının Etiketleme: (655nm) 100 ul PBS olmayan hedefli kuantum noktaları 15μl kan damarları, görselleştirmek için seyreltilir ve görüntüleme önce fare 10-30 dakika içinde intravenöz olarak enjekte edilir. </li><li> Anestezi: <ol><li> Veteriner inhalasyon anestezi sistemi (Noble Sağlık Hizmetleri A.Ş.) indüksiyon odasında fare yerleştirin. </li><li> Buharlaştırıcı İsofluranın% 3-4 ve% 100 oksijen taşıyıcı gaz akış hızı ayarlayın ~ 1 L / dk. Arka ayak tutam refleks kaybı kadar yanlışlıkla anestezi doz aşımı önlemek için, hayvan yakından izlemek. </li><li> Anestezi 1.5 muhafaza görüntüleme sırasında gerektiğinde solunum bozulmaları en aza indirmek için ayarlanmış ~ 2% İsofluranın ve ince. Farenin solunum hızı, anestezi yeterli bir düzlemde sigortalamak için görüntüleme oturum boyunca dikkatle izlenmelidir. </li><li> Puralube veteriner merhem gözlerin kurumasını önlemek için uygulanır. </li><li> Fare düzenlenmiş 36 yerleştirilir ° C ısıtma yastığı (Braintree Bilimsel) enjeksiyonları ve görüntüleme sırasında ve hipotermi önlemek için. </li><li> Dehidratasyonu önlemek için, fare 100 ul PBS sc her 1-2 saatte bir verilir. </li></ol></li><li> Inflamatuar bir uyarana teslim lökosit işe ikna etmek için: <ol><li> Cerrahi evre ve% 70 etanol ile Kapkaççı çubukla fare yerleştirin. </li><li> Insülin şırınga, iğne, enjeksiyon teslim derinliği hassas kolaylaştırmak ve geliştirmek için ~ 90 derece eğim yakın bir bükün. </li><li> 1μl PBS içinde seyreltilmesi bioparticles E.coli 0.5 mg şırınga yükleyin. Bu kolay damlacık yük yapmak mikrofuge&#39;de tüp kapağı yapılabilir. </li><li> Kavisli bir forseps ile parmak tutun ve derinin hemen altına kadar eğim yukarı bakacak şekilde iğne ile deri yoluyla yavaşça yerleştirin. </li><li> Yavaş yavaş enjekte edilir ve herhangi bir geri boşaltma önlemek için 5 saniye boyunca basılı tutun. </li></ol></li></ol> 2. Görüntüleme sahne kurulumu: Görüntüleme platformu merkezi üzerinden kesilerek yuvarlak bir delik olan bir alüminyum levha oluşur. Büyük bir kapak cam plaka ve lastik bir O-ring, arka pençe karşılamak için sıvı geçirmez bir gömme odasının oluşturmak için üstüne yapıştırılmış altına yapıştırılmış. 36 ° C plaka üstüne bağlanmış iki ısıtma elemanları ile alüminyum levha ısıtılır. <ol class="lalpha"><li> Önceden ısıtılmış görüntüleme sahnede fare koyun ve 36 ° C ısıtıcı kullanarak farenin çekirdek vücut ısısını korumak. </li><li> Superglue ince bir film tabakası ile görüntüleme odasının coverglass pençe sabitleyin. </li><li> Tutkal iki defa herhangi bir kayan bit kaldırmak ve daha sonra odanın taze PBS (36 ° C önceden ısıtılmış) ile doldurmak için pençe yıkayın. </li></ol> Fare hayvan canlılığı oluşturan 4 saate kadar görüntülenebilir. Ayak yavaşça küçük miktarda aseton ve uzunlamasına görüntüleme çalışmaları için yeniden canlandı fareler ile serbest bırakılabilir. Ancak, bu durumda, biz görüntüleme süresi &lt;2 saat tavsiye ve anestezi doz aşımı ya da dehidratasyon riskini en aza indirmek için oturumlar arasında 24-48 saat bekliyor. 3. Imaging Acquisition Bu protokolde kullanılan iki foton mikroskobu (2P mikroskop) dik bir mikroskop oluşan özel bir dahili video hızı dual lazer sistemi. Sistem iki Ti ile donatılmıştır: Sapphire lazerler, dört baş-3 ve 4 kanal aynı anda satın almalar ve 20x suya daldırma hedefi (Olympus, 20x/0.95 NA) Bi-alkali Proje Yönetim Ekipleri. <ol class="lalpha"><li> 890-900nm safir lazer (Tutarlı A.Ş.): Chameleon XR Ti ayarlayın. </li><li> , Mavi (&lt;480 nm, ikinci harmonik üretimi sinyali), yeşil (480-560nm, LYSM eGFP pozitif nötrofil) ve kırmızı (&gt; 560 nm, kuantum nokta etiketli kan damarlarının üç kanal ayrı 480nm ve 560nm dikroik aynalar (SemRoc) kullanın. .) Alternatif olarak, 560nm filtre E. ayırt etmek için bir 570nm filtre ile değiştirilebilir 655nm kuantum noktaları (kırmızı derin görünür) coli bioparticles (576nm bioparticles turuncu görünecektir). </li><li> PBS içine dikkatlice amacı alt ve ayak bir gemi odaklanmak. </li><li> Görüntü elde etme hızı, video-oranı (30f/sec) işletme ve kollajen doku işareti olarak, hızlı bir şekilde ilgi (tek bir kan damarı bulmak için (video oranının toplama hızı) doku anket kaynaklanan ikinci harmonik sinyali kullanılarak ile görünür LYSM eGFP nötrofiller). </li><li> Proje Yönetim Ekipleri (renk ayrımı optimize etmek ve arka plan üzerinde yeterli sinyal elde etmek için gerekli lazer miktarını en aza indirmek için kazanç ayarlayın.dikkatli bir görüntü doyurmak için değil!). </li><li> Çıkarlarının alan bölgede bulunan sonra, zaman atlamalı görüntüleme başlar. Time-lapse görüntüleme, iki farklı zamansal çözünürlükleri ile yapılabilir. , Görüntüleme hücre yuvarlanma ve yapışma için ve kan damarları içinde gerçek zamanlı olarak, tek bir z-düzlemi 30f/sec kazanmak. Parankim hücre ekstravazasyonu ve göç belgeleyen için 3B zaman atlamalı görüntüleme gerçekleştirin. Tipik bir satın alma protokolü, her Z-adım için ortalama 15 kare ve 30 saniye aralıklarla 200-225 50μm z adımları bir birim için 21 sıralı 2.5μm elde oluşacaktır. </li><li> Sonra veri dosyaları laboratuar sunucu üzerinde saklanır, Imaris veya Volocity yazılımı 2 izleme çok boyutlu render ve hücre, 3 gerçekleştirmek için kullanılabilir. </li></ol> 4. Temsilcisi Sonuçlar Nötrofil hayal 1.Real-time (30f/sec) 200x damarlar boyunca yuvarlanan. Listeria monocytogenes ile enfeksiyon sonrası kırmızı renkte gösterilen 10mins hakkında damarların endotel boyunca zaman atlamalı görüntüleme haddeleme, yeşil, nötrofil gösterir. Bağ dokuda kollajen lifler mavi görünür. <a href="http://www.jove.com/files/ftp_upload/2062/2062_Miller_Movie1.mov">videoyu görmek için buraya tıklayınız.</a> 200x nötrofil göçünü dinamikleri 2.Time hızlandırılmış 3D görüntüleme. Tipik nötrofil göçünü iltihaplı doku dolaşım ve göç gösterilmiştir. <a href="http://www.jove.com/files/ftp_upload/2062/2062_Miller_Movie2.mov">videoyu görmek için buraya tıklayınız.</a>

<ol>
	<li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Zinselmeyer, B. H., Lynch, J. N., Zhang, X., Aoshi, T., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+two-photon+imaging+of+mouse+footpad+-+a+promising+model+for+studying+leukocyte+recruitment+dynamics+during+inflammation.">Video-rate two-photon imaging of mouse footpad - a promising model for studying leukocyte recruitment dynamics during inflammation.</a> Inflamm Res. 57, 93-96 (2008).</li><li>Zinselmeyer, B. H., Dempster, J., Wokosin, D. L., Cannon, J. J., Pless, R., Parker, I., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16.+Two-photon+microscopy+and+multidimensional+analysis+of+cell+dynamics.">Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics.</a> Methods Enzymol. 461, 349-378 (2009).</li><li>Gray, D. F., Jennings, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergy+in+experimental+mouse+tuberculosis.">Allergy in experimental mouse tuberculosis.</a> Am Rev Tuberc. 72, 171-195 (1955).</li><li>Lin, A., Loughman, J. A., Zinselmeyer, B. H., Miller, M. J., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+S+inhibits+neutrophil+recruitment+during+the+early+stages+of+Streptococcus+pyogenes+infection.+Infect+Immun.">Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection. Infect Immun.</a> 77, 5190-5201 (2009).</li><li>Smith, C. J., Zhang, Y., Koboldt, C. M., Muhammad, J., Zweifel, B. S., Shaffer, A., Talley, J. J., Masferrer, J. L., Seibert, K., Isakson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+analysis+of+cyclooxygenase-1+in+inflammation.">Pharmacological analysis of cyclooxygenase-1 in inflammation.</a> Proc Natl Acad Sci U S A. 95, 13313-13318 (1998).</li><li>Wipke, B. T., Allen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+neutrophils+in+the+initiation+and+progression+of+a+murine+model+of+rheumatoid+arthritis.">Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis.</a> J Immunol. 167, 1601-1608 (2001).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc Res. 5, 384-394 (1973).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">in vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li><li>Miller, M. J., Wei, S. H., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy.">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc Natl Acad Sci U S A. 100, 2604-2609 (2003).</li></ol>

St Louis Washington Üniversitesi Hayvan Araştırmaları Komitesi tarafından onaylanan protokollere göre Tüm deneyler yapıldı.

Bu video protokol enflamasyona yanıt olarak, non-invaziv lökosit alımı 2P görüntüleme için prosedürler göstermektedir. Kapkaççı gibi alerji, enfeksiyon ve otoimmün hastalığı 4-7 gibi inflamasyon çalışmaları klasik bir fizyolojik sitesidir. 2P görüntüleme, yuvarlanma ve kan damarlarında effektör fonksiyonu siteleri için kemotaksis yapışma, lökosit kaçakçılığı yollar farklı adımları ayrıntılı bir resim sağlar. Biz lökosit işe farklı tipt...

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<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Butler Animal Health Supply</td>
<td>029405</td>
<td>&nbsp;</td>
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<td>655nm non-targeted quantum dots</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Q21021MP</td>
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<td>Tetramathylrhodamine conjugated E. coli BioParticles </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>E2862</td>
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<td>Listeria monocytogenes (Lm)</td>
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non-invasive imaging of leukocyte homing and migration in vivo

İki foton (2P) mikroskobu, yüksek çözünürlüklü bir görüntüleme tekniği geniş biyologlar tarafından adapte edilmiştir. 2P mikroskopi değeri dokulara içinde ve canlı farelerde hücre davranışları ile ilgili zengin Spatiotemporal bilgi sağlar. Lökosit işe host enfeksiyona karşı savunma ve ne zaman kontrolsüz önemli bir rol oynar, inflamatuar ve otoimmün hastalıklar için katkıda bulunabilir. Hücreler, ışık saçılması dokular içinde derin bulunan gemiler içinde hızla hareket ediyor bu yana, in vivo olarak lökosit işe incelenmesi teknik olarak çok zordur. Bugüne kadar, en intravital görüntüleme çalışmaları, kan damarları ve dokuların ortaya çıkarmak için cerrahi hazırlık gerektirir. Cerrahi kendisi tarafından indüklenen doku hasarı ve inflamasyon önlemek için, burada, biz fare Kapkaççı ve bilek lökosit ticaretini incelemek için kullanılabilecek bir tek hücreli non-invaziv görüntüleme yaklaşımı tanımlar. Biz, bizim 2P görüntüleme hazırlık teknik yönlerini tartışmak ve yürütme ve veri toplama için kurulmuş ilk tipik bir deney yoluyla okuyucu yürümek.

Watch this Scientific Journal Video about Non-invasive Imaging of Leukocyte Homing and Migration in vivo at JoVE.com

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Burada, fare Kapkaççı lökosit homing çalışmada non-invaziv bir iki foton (2P) mikroskopi yaklaşımı açıklar. Biz, bizim doku görüntüleme hazırlık teknik yönlerini tartışmak ve yürütme ve veri toplama için kurulmuş ilk tipik bir deney yoluyla okuyucu yürümek.

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 ...

non-invasive-imaging-of-leukocyte-homing-and-migration-in-vivo

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

12.7K Görüntüleme.  Washington University in St. Louis. Burada, fare Kapkaççı lökosit homing çalışmada non-invaziv bir iki foton (2P) mikroskopi yaklaşımı açıklar. Biz, bizim doku görüntüleme hazırlık teknik yönlerini tartışmak ve yürütme ve veri toplama için kurulmuş ilk tipik bir deney yoluyla okuyucu yürümek.

Video: Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

Gut-tropik T Hücre Göç Eğitim Rekabetçi Homing Tahliller

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

İmmünoloji ve Enfeksiyon

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Vasküler Endotel Yüksek Lumen Basıncı Görüntüleme Lökosit Adezyon

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

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.

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 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 ...

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

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

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

Farelerde mesenteriumda Damarlardaki intravital Mikroskopi Lökosit-endotelyal ve Trombosit-lökosit Etkileşimleri

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

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Iltihaplı Dermisinde içinde görüntüleme CD4 + T hücre Geçiş Göç

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...