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, pH 6.8, 5 mM NaCl) metilen mavisi ile (0.3 ug / ml nihai E3 ortam 150 ml içeren 15 cm&#39;lik bir petri tabaklarında 120 yumurta inkübe 12/12 açık / koyu fotoperiyodun ile 33 ° C inkübatör konsantrasyon). </li><li> E3 + propiltiyourasil&#39;i (1-fenil-2-tioüre, 10 ug / ml nihai konsantrasyon) ile 6 saat sonra ortam yerine ve ölü yumurta çıkarın. </li><li> 2 gün sonra, taze E3 + propiltiyourasil&#39;i Ortamı alışverişi, 33 ° C&#39;de 4 gün boyunca inkübe edilir. </li></ol> 2. içindejection mikro-iğne hazırlanması <ol><li> Aşağıdaki ayarlarla bir iğne çektirmenin kullanarak bir mikro-iğne içine; borosilikat kılcal (ID 0.69 mm OD 1.2 mm) çekin (550 Heat, 0 çekin, 130 Velocity 110 Saat). NOT: Ayarlar filamanın değişecektir (Şekil 1) benzer şekilli mikro-iğneler elde etmek filament ve yeni bir filaman takıldığında yeniden ayarlanması gerekir. </li><li> 0.4 um filtreden fosfat tamponlu salin 3 ul mikro-iğne ile doldurun (; 137 mM NaCI; 2.7 mM KCI, PBS, 10 mM, Na 2 HPO 4; 1,8 mM KH 2 PO 4, 7.4 pH) 20 ul Microloader pipet kullanılarak ucu. </li><li> Bir basınçlı enjeksiyon sistemine bağlı bir micromanipulator bir mikropipet tutucu mikro-iğne ayarlayın. 30 PSI enjeksiyon basıncını ve 30 msn de darbe süresini ayarlayın. </li><li> Iğne 1/5 su içinde olana kadar damıtılmış su ile bir petri kabı kullanarak, mikro iğne düşük. Mikro-iğne, wi Klipsu seviyesinin hemen altında ince cımbız th. Sudan mikro-iğne getir ve bir bolus görünene kadar pedalına birkaç kez itin. </li><li> Çeşidi bir damla bir daldırma yağı ile katmanlı bir hemositometrede mikro-iğne düşürerek bolus çapını ölçün. İstenilen hacim elde etmek için darbe süresi ayarlayın (Tablo 1). </li><li> Yağda stabil kalır (bolus hacmini artırır eğer basıncı düşürmek ve Kapsül hacmi azalırsa backpressure artırmak) için bolus için backpressure ayarlayın. Her mikro-iğne darbe süresini kaydedin. </li><li> Bir Microloader pipet kullanarak aspire mikro-iğne PBS çıkarın ve enjektör geri yerleştirerek ve sürekli darbe anahtarı çevirerek sol-over PBS sınırdışı. Her mikro-iğne Rezervasyon. </li></ol> 3. Candida Hazırlık <ol><li> Streak Candida albicans kodon-optimize dTomato, GFP, BFP, EOS veya Far ile transformeBir maya peptine dekstroz (YPD) agar (10 g / L maya ekstresi, 20 g / L pepton üzerine steril ahşap takoz kullanılarak donmuş stoktan (-80 ° C hazır% 25 gliserol), kırmızı, 20 g / L dekstroz , 20 g / L ağar; otoklava Petri kapları içinde 25 ml boşaltılmıştır) ve 30 ° C de bir gece boyunca inkübe edilir. </li><li> Steril bir ahşap takoz kullanılarak bir koloni almak ve YPD bir sıvı (10 g / L maya ekstresi, 20 g / L pepton, 20 g / L dekstroz, 5 ml kadar inoküle, Otoklavdan geçirilen ve aseptik 16 x 150 mm cam kültürü, 5 ml bölünebilir borular). </li><li> 60 rpm&#39;de bir silindir tambur içinde 30 ° C&#39;de gece boyunca inkübe edin. </li><li> C. 1 ml toplayın 1.7 ml steril santrifüj tüpü içine albicans kültürü ve transferi. </li><li> Sn bir çift için 5.000 xg&#39;de santrifüj. Süpernatant boşaltın ve iyice steril PBS, girdap 1 ml ekleyin. Iki kez bu işlemi tekrarlayın. </li><li> Steril PBS içinde 1000: 1 ml stok 1 seyreltilmesi ile bir hemositometre kullanılarak kolonileri sayın. </li></ol> 4. Zebra balığı EnjekteSwimbladder <ol><li> Bir 33 ° C kuluçka makinesi içinde, 30 dakika için bir enjeksiyon çanak (% 2 agaroz) ısıtın. </li><li> İstenen zaman, herhangi bir zebrafish aspire olmadan 25 mi pipet kullanılarak enjekte balık içeren 15 cm Petri için E3 + propiltiyourasil&#39;i, 150 ml, 100 çıkarın. </li><li> Geri kalan, 50 ml ortam (nihai konsantrasyon 200 mg / ml) bir 4 mg / ml tampon tricaine metan sülfonat stok (TR) 2 ml ekleyerek zebrabalıkları dpf 4 anestezisi ve 15 dakika boyunca bekleyin. </li><li> Bir mikroskop altında, şişirilmiş bir statik taşıma ile balık seçin. Balık aynı zamanda MPO nötrofil dağıtımı, homojen fenotip için o anda taranabilir: GFP çizgi mikroskop diseksiyon bir Epifloresans kullanarak. </li><li> C seyreltin orijinal konsantrasyon albicans stok 1.5 x 10 7 koloni PBS ml (cfu / ml) oluşturan üniteler için (adım 3.8). </li><li> Plastik bir transfer borusu kullanılarak enjeksiyon çanak üzerine 30 zebrafish aktarıntte ve aşırı ortamı çıkarın. </li><li> Bir balıkçı tel aracı (0.012 inç çaplı olta borosilikat kılcal içine süper yapıştırılmış) kullanarak, dikey enjeksiyon çanak tutarak 10 balık, 3 çizgiler yapmak ve dikey balık yönlendirmek. </li><li> Fazla ortamı çıkarın. </li><li> İyice 1.5 x 10 7 cfu ile tüp vorteks / mL C albicans arasından seçilir. </li><li> C 3 ul bir mikro-iğne doldurun albicans, yeni bir Microloader pipet kullanarak ve uygun ayara darbe süresini ayarlamak (adım 2.9 bakınız). </li><li> Swimbladder (Şekil 3A) arkasına doğru amaçlayan, mikro-iğne ve balık kafası arasındaki 20 ° lik bir açı yapmak için enjeksiyon çanak döndürün. </li><li> , Statik taşıma içine mikro-iğne itin iğne deliği lümen olduğundan emin yapma, ve bir kez pedalına basın. </li><li> Her balık için tekrarlayın ve E3 bir çanak sel bir kurtarma çanak (25 ml E3 + PTU) transferd kurtarma çanak içine boşalma. </li><li> Enjeksiyon çanak başka 30 balık aktarın. </li><li> İyice ile dolu yeni bir mikro-iğne ile enjeksiyon tekrarlayın C vortekslendi albicans arasından seçilir. </li><li> Gerekirse kirlenmesini önlemek ve ayrı bir kurtarma çanak (adım 4.10) aktarmak için farklı bir enjeksiyon çanak kullanarak, PBS kontrol enjekte edilerek bitirin. </li></ol> 5. İstenilen İnokulum için Balık tarama <ol><li> Çözülene kadar mikrodalga ve 37 ° C&#39;ye kadar soğutularak kaynatma, E3 40 ml 0.4% agaroz çözeltisi (LMA) düşük eriyik 160 mg ekleyerek, düşük erime sıcaklığı olan agaroz için 40 mi hazırlayın. Bir kez soğutulduğunda TR (nihai konsantrasyon 200 mg / ml) 400 ul ekle. </li><li> (25 mL, E3 + propiltiyourasil&#39;i, nihai konsantrasyon 200 mg / ml içine TR sadece 1 ml ile adım 4.3) de anlatıldığı gibi, 30 dakika sonrası nekahattan sonra, balık uyuşturan. </li><li> Bir kilo tekne LMA 2 ml (mümkün olduğunca az medya ile) Transfer 30 balık. </li><li> Aspire ayrı bir transfer pipeti ile balık ve sadece 10 x 6 merkez kuyu kullanılarak, 96 çukurlu bir görüntüleme plaka içine LMA&#39;nın 1 damla balık plaka. </li><li> Ekranın tüm balık görüntüleme plakasına yüklenir kadar tekrarlayın. </li><li> Ortam, 5 dakika boyunca oda sıcaklığında katılaşmaya edelim. </li><li> Onlar cam alt ile temas halinde emin, kendi tarafında balık yerleştirin. </li><li> Bir ters Epifloresans / konfokal mikroskop ve uygun filtre üzerinde 20X objektif kullanılarak, C sayısını kaydetmek Her balığın statik taşıma albicans maya hücreleri. </li><li> , Statik taşıma 15-25 maya hücreleri ile balık (veya arzu edilen inokulum) Select ötenazi seçili olmayan balık (E3 TR 800 ug / ml) tarafından E3 + propiltiyourasil&#39;i 50 ml derin Petri kabı seçilen balık dönüş görüntüleme plaka seçilen kuyulara E3 3 damla ekleyerek ve plastik transfer pipet ile balık aspire. </li><li> İstenen süre için 33 ° C&#39;de inkübe edin. </li></ol> 6. Balık Sonrası tarama Görüntüleme <ol><li> İstediğiniz anda, görüntü balık içeren derin Petri kabı E3 + PTU medya 50 ml 25 kaldırın. </li><li> (25 mi, E3 + propiltiyourasil&#39;i kalan, nihai konsantrasyon 200 mg / ml içine TR sadece 1 ml ile adım 4.3) de anlatıldığı gibi balık anestezi uygulayın. </li><li> Uygun bir şekilde 96 oyuklu görüntüleme levhasının ve yerine balık Plate (5,3-5,7 adım). </li><li> Uygun lazerler ve emisyon filtreleri kullanarak konfokal mikroskobu ile görüntü. İlk diferansiyel girişim kontrast (DIC) kapsamında 4X objektif kullanarak balık odaklama ve 2 mikro saniye / piksel hızı ve 1024 x 600 piksel en boy oranı ile tarama modunda konfokal lazerler ile 20X objektif kullanarak ilgi bölgenin görüntülerini elde. 3 kare Kalman filtresi modunu uygulayarak, 1 mikron adım boyutu ile Z-yığınlar edinin. </li><li> 50 ml E3 + PTU veya individua içine derin bir Petri kabı dönl kuyuları (24 bölümlü kültür çanağı, 3 mi E3 + PTU) ile 33 ° C&#39;de inkübe edin. </li></ol> 7. swimbladder Dissecting <ol><li> Yüksek vakum yağ ile bir 10 ml şırınga doldurun. </li><li> % 2&#39;lik bir LMA (agaroz E3 8 ml düşük erime 160 mg adım 5.3) hazırlayın. </li><li> İstediğiniz anda, balık (adım 5.2) uyuşturan. </li><li> Transferi 10 E3, 1 ml ile 1.7 ml santrifüj tüpüne balık ve santrifüj tüpüne 4 mg / ml &#39;Tricaine (nihai konsantrasyon 800 mg / ml) 200 ul ilave edildi ve oda sıcaklığında 15 dakika boyunca inkübe edilerek euthanize. </li><li> Bir mikroslayd (75 x 25 mm, Şekil 2) 1 cm arayla, bir meydanda vakum gres 4 damla koyarak bir görüntüleme cam slayt hazırlayın. </li><li> Meydanın ortasında% 2 LMA 2 damla yerleştirin. </li><li> Bir mikroskop altında ayrı bir cam slayt bir balık yerleştirin ve kalp atışlarımı durduğundan emin olun, aşırı su çıkarmak. </li><lİnce cımbız kullanarak i&gt; sol cımbız ile sabit bir pozisyonda kafa korumak ve doğru cımbız (Şekil 4A) ile ön gut aşağı çekerek swimbladder teşrih. </li><li> Sindirim sisteminden ayıran, sol cımbız ile pnömatik kanal tarafından swimbladder Pick up ve katılaşır önce% 2 LMA merkezine yerleştirin. </li><li> Görüntüleme cam slayt üstüne bir kapak kayma (18 x 18 mm) yerleştirin ve vakum gres yanı sıra, LMA (Şekil 2) dokunana kadar itin. </li><li> Diseksiyon 10 dakika içinde Resim aşama 6.4&#39;te tarif edildiği gibi. Adımı tekrarlayın 7,7-7,11 diğer balık. </li></ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>1.7 ml tubes</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Deep Petri dishes</td><td>Fisher Scientific</td><td>89107-632</td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Yeast Extract</td><td>VWR Scientific</td><td>90000-726</td><td></td></tr><tr><td>Peptone</td><td>VWR Scientific</td><td>90000-264</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Agar</td><td>VWR Scientific</td><td>90000-760</td><td></td></tr><tr><td>Fine tweezers (Dumont Dumoxel #5)</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Wooden dowels</td><td>VWR Scientific</td><td>10805-018</td><td></td></tr><tr><td>Low melt agarose</td><td>VWR Scientific</td><td>12001-722</td><td></td></tr><tr><td>Flaming Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Borosilicate capillary</td><td>Sutter Instruments</td><td>BF120-69-10</td><td></td></tr><tr><td>MPPI-3 injection system</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr><tr><td>Back pressure unit</td><td>Applied Scientific Instrumentation</td><td>BPU</td><td></td></tr><tr><td>Micropipette holder kit</td><td>Applied Scientific Instrumentation</td><td>MPIP</td><td></td></tr><tr><td>Foot switch</td><td>Applied Scientific Instrumentation</td><td>FSW</td><td></td></tr><tr><td>Micromanipulator</td><td>Applied Scientific Instrumentation</td><td>MM33</td><td></td></tr><tr><td>Magnetic base</td><td>Applied Scientific Instrumentation</td><td>Magnetic Base</td><td></td></tr><tr><td>Tricaine methane sulfonate</td><td>Western Chemical Inc.</td><td>MS-222</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ61 top SZX-ILLB2-100 base</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>IX-81 with FV-1000 laser scanning confocal system</td><td></td></tr><tr><td>20X microscope objective</td><td>Olympus</td><td>UPlanSApo 20x/0.75</td><td></td></tr><tr><td>Roller drum</td><td>New Brunswick Scientific</td><td>TC-7</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Glass culture tubes (16 x 150 mm)</td><td>VWR Scientific</td><td>60825-435</td><td></td></tr><tr><td>NaCl</td><td>VWR Scientific</td><td>BDH4534-500GP</td><td></td></tr><tr><td>KCl</td><td>VWR Scientific</td><td>BDH4532-500GP</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR Scientific</td><td>BDH0246-500GP</td><td></td></tr><tr><td>HEPES (Corning)</td><td>VWR Scientific</td><td>BDH4520-500GP</td><td></td></tr><tr><td>Children clay (Play-Doh)</td><td>Hasbro</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>C69-500</td><td></td></tr><tr><td>Methylene blue</td><td>VWR Scientific</td><td>VW6276-0</td><td></td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629-10G</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Hemocytometer (Hausser scientific)</td><td>VWR Scientific</td><td>15170-172</td><td></td></tr><tr><td>Type A immersion oil</td><td>Blue Marble Products</td><td>51935</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Vortex Genie</td><td>VWR Scientific</td><td>14216-184</td><td></td></tr><tr><td>Agarose (Lonza)</td><td>VWR Scientific</td><td>12001-870</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>Fishing wire</td><td>Stren</td><td></td><td></td></tr><tr><td>96 well imaging plate (Sensoplate)</td><td>Greiner Bio-One</td><td>655892</td><td></td></tr><tr><td>High vacuum grease (Dow Corning)</td><td>VWR Scientific</td><td>59344-055</td><td></td></tr><tr><td>Microslide (25 x 75 mm)</td><td>VWR Scientific</td><td>48300-025</td><td></td></tr><tr><td>Cover slips (18 x 18 mm), No 1.5</td><td>VWR Scientific</td><td>48366-045</td><td></td></tr><tr><td>15 cm Petri dish (Olympus plastics)</td><td>Genesee Scientific</td><td>32-106</td><td></td></tr><tr><td>Glycerol (EMD chemicals)</td><td>VWR Scientific</td><td>EMGX0185-5</td><td></td></tr><tr><td>24-well culture dish (Olympus plastics)</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>Weight boats (8.9 cm)</td><td>VWR Scientific</td><td>89106-766</td><td></td></tr></tbody></table>

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 ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.1K Görüntüleme.  University of Maine. 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.

Video: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

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 ...

İmmünoloji ve Enfeksiyon

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Kuruluş, larva Zebra balığı bir hayvan modeli araştırmak Trypanosoma cruzi hareketliliği In Vivo olarak

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Gelişim Biyolojisi

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Görüntüleme Patogenez ve Cording Deşifre ve<em&gt; Mycobacterium abscessus</em&gt; Zebra balığı Embriyolar

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Clostridioides difficile için Larva Zebrabalığı Enfeksiyon Modeli nin Geliştirilmesi

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

İnsan İnfluenza A Virüs Enfeksiyonları Zebra balığı Modeller Kullanılarak Antiviral İlaçlar Ekran ve Host Bağışıklık Hücre Yanıtları karakterize etmek

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Tüberküloz Mycobacterium marinum enfekte yetişkin zebra balığı içinde modelleme

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

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

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Uyuşturucu Yüklü Lipozomenjekte ederek Larva Zebra balığı Makrofajlarına Yönelik İlaçların Hedeflenmesi

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Konakçı-Patojen Etkileşimlerinin Analizi için Zebra Balığı Larvalarının Aspergillus Sporları ile Enfeksiyonu

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Nöroinflamasyon ve Nörotoksisiteyi Değerlendirmek için Larva Zebra Balıklarına Lipopolisakkaritin Beyin Ventriküler Mikroenjeksiyonları

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Nörobilim

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Source: Thrikawala, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2020)
The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

Mantar Patojeni ile Enfekte Zebra Balığı Larvalarını Görselleştirmek için Bir zWEDGI Tekniği

1. Preparation of&#160;Aspergillus&#160;spores for injection

	From an&#160;Aspergillus&#160;spore suspension, calculate the volume needed to obtain 1 x ...