Name: modeling mucosal candidiasis in larval zebrafish by swimbladder injection
Uploaded: 2014-11-27T14:00:00
Description: Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Yazarlar cömertçe α-katenin sağlamak için Dr. Le Trinh ve Dr. Tobin teşekkür: sitrin balık hattı ve Bill Jackman bize onun laboratuarında filme yapmak için izin için. Yazarlar fon kaynaklarını Ulusal Sağlık Enstitüleri (Hibeler 5P20RR016463, 8P20GM103423 ve R15AI094406) ve USDA kabul (Proje # ME0-H-1-00517-13). Bu yazının ana Tarım ve Ormancılık Deney İstasyonu yayın sayısı 3371 olarak yayınlanmıştır.

NOT: Tüm Zebra balığı bakım protokolleri ve deneyler Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) protokolü A2012-11-03 altında NIH kurallarına uygun olarak yapıldı. 4 Gün Post Döllenme 1. Zebra balığı Yetiştirme <ol><li> Başka bir video 34 &#39;de gösterildiği gibi, ilk 3 saat sonra döllenme içinde, AB zebrafish veya başka bir transjenik çizgiler toplayın. </li><li> (0.17 mM KCI, 0.33 mM CaCl2, 0,33 mM MgCl2, 2 mM HEPES,...

Swimbladder mikroenjeksiyon hastalığı modeli Gelişmeler ve sınırlamaları Burada sunulan model Gratacap et al mukozal kandidiyaz daldırma modelinin bir uzantısıdır (2013).; Bu kontrollü bir enfeksiyon zaman avantaj, yüksek ölçüde tekrarlanabilir enfeksiyon dozu ve bu nedenle geliştirilmiş verimlilik ekler. Biz burada non-invaziv ayrıntılı olarak enfeksiyon dinamikleri zamansal belgeleri yanı sıra statik taşıma yüksek çözünürlük ex vivo g?...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

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			<td height="21" style="height:21px;">1.7 mL tubes</td>
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			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
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			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
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			<td>VWR Scientific</td>
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			<td>VWR Scientific</td>
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			<td>VWR Scientific</td>
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			<td>VWR Scientific</td>
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modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Mukozal patojenlere karşı savunma Erken bir epitel bariyer ve doğuştan gelen bağışıklık hücreleri, hem oluşur. Her iki immunocompetency ve bunların interkom, enfeksiyonlara karşı korunması için çok önemlidir. Bir patojen ile epitel ve doğuştan gelen bağışıklık hücreleri etkileşimleri en karmaşık davranış zaman ve mekan içinde izlerken vivo, incelenmiştir. Ancak, mevcut modeller mukozal düzeyinde patojenler ile savaş kolay uzay-zamansal görüntüleme için izin vermez. Burada geliştirilen model çocuk zebrabalıkları statik taşıma içine mantar patojeni, Candida albicans, doğrudan enjeksiyon sureti ile bir mukozal infeksiyonu oluşturur. Ortaya çıkan enfeksiyon mukozal hastalık gelişimi boyunca epitel ve doğuştan gelen bağışıklık hücre davranışının yüksek çözünürlüklü görüntüleme sağlar. Bu yöntemin çok yönlülük ph yol açan bağışıklık olaylar ayrıntılı dizisini prob konağın sorgulama için izin veriragocyte işe özel hücre tipleri ve koruma moleküler yolların rollerini incelemek ve. Buna ek olarak, immün bir fonksiyonu olarak patojen davranışı floresan protein ifade C ile eş zamanlı olarak görüntülenebilir albicans arasından seçilir. Konak-patojen etkileşimi artmış uzaysal çözünürlüğü açıklanan hızlı swimbladder diseksiyon tekniği kullanılarak da mümkündür. Burada anlatılan mukozal enfeksiyonu modeli mukozal kandidiazis çalışma için değerli bir araç yapma, basit ve son derece tekrarlanabilir. Bu sistem, aynı zamanda, normal epitel yüzeyleri boyunca enfekte mikobakteriyel bakteriyel veya viral mikropların gibi diğer mukozal patojenlerin geniş çapta çevrilebilir olabilir.

Posterior statik taşıma içinde Mikroenjeksiyon Burada sunulan deneysel yöntem C tutarlı bir doz enjeksiyon açıklar zebrabalıkları dpf 4 statik taşıma albicans maya hücreleri. Daldırma modeli ile Önceki iş önerdiği C swimbladder immün yanıt Candida albicans, memeli mukozal kandidiyazis 32 benzerdir. Burada daha basit, tekrarlanabilir ve hızlı bir modifiye enfeksiyon yöntemi göstermektedir; zebrabalıkları bir kaç yüz enjekt...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

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.

Swimbladder Enjeksiyon ile Larva Zebra balığı Mukozal Candidiazis Modelleme

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

Research

JoVE Journal

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

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

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

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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 ...

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This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

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Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

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Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...