Summary

Неинвазивная визуализация диссеминированный кандидоз в Личинки данио рерио

Published: July 30, 2012
doi:

Summary

Бурное развитие, малый размер и прозрачность данио огромные преимущества для изучения иммунной контроль инфекции<sup> 1-4</sup>. Здесь мы покажем методы заражения личинки данио использованием возбудителей грибковых<em> Candida Albicans</em> По микроинъекции, методология недавно использовали, чтобы обвинить фагоцитарную активность NADPH оксидазы в управлении грибковых диморфизм<sup> 5</sup>.

Abstract

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.

Protocol

Все данио протоколы ухода и эксперименты проводились в условиях институционального ухода и использования животных комитета (IACUC) протокол A2009-11-01. 1. Морфолино и личинок посуды инъекций Экспериментальные продолжительность: * (10-15 минут) <p class="jove_co…

Discussion

Данио методом микроинъекции, представленные здесь отличается от Gutzman и др. 34. В том, что здесь мы демонстрируем инъекции через слуховым пузырьком в задний мозг желудочка 36 до 48 личинок HPF. Метод описан позволяет последовательное введение 10-15 дрожжи в задний мозг желудочка с ум?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Авторы хотели бы поблагодарить лабораторию доктора Кэрол Ким микроинъекции для обучения, Кларисса Генри совет по ускорению развития эмбриона и использования оборудования, и Натан Лоусон за вклад fli1: EGFP рыбы. Мы благодарим членов Уилер лаборатории и Шон стены на критическое чтение рукописи. Мы также хотели бы поблагодарить Марка Nilan для ухода рыбы и советами, и Райан Phennicie и Кристин Габора для технических консультаций по этому проекту. Эта работа финансировалась ассистентом MAFES исследования К. Brothers, MAFES Hatch грант E08913-08, и награда NIH NCRR P20RR016463 Р. Уилер.

Materials

Name of the reagent Company Catalog number Comments (optional)
Spawning tanks Aquatic habitats  2L  
1.7 mL tubes Axygen MCT-175-C
Instant Ocean Fisher Scientific S17957C  
Extra deep Petri dishes Fisher Scientific 08-757-11Z  
Standard Petri dishes VWR Scientific 25384-302
Transfer pipettes Fisher Scientific 13-711-7M  
Yeast Extract VWR Scientific 90000-726  
Peptone VWR Scientific 90000-264  
Dextrose Fisher Scientific D16-1  
Agar VWR Scientific 90000-760  
Disposable Hemocytometer VWR Scientific 82030-468  
Phosphate Buffered Saline VWR Scientific 12001-986  
Dumont Dumoxel Tweezers VWR Scientific 100501-806  
Wooden Dowels VWR Scientific 10805-018
KimWipes VWR Scientific 300053-964
Low Melt Agarose VWR Scientific 12001-722  
Agarose for injection dishes VWR Scientific 12002-102
Flaming Brown Micropipette Puller Sutter Instruments P-97
Hollow glass rods Sutter Instruments BF120-69-10 For glass rods smooth glass by heating over bunsen burner 
Pipette Storage Box Sutter Instruments BX10
MPPI-3 Injection system Applied Scientific Instrumentation MPPI-3
Back Pressure Unit Applied Scientific Instrumentation BPU  
Micropipette Holder kit Applied Scientific Instrumentation MPIP  
Foot Switch Applied Scientific Instrumentation FSW  
Micromanipulator Applied Scientific Instrumentation MM33  
Magnetic Base Applied Scientific Instrumentation Magnetic Base  
Tricaine methane sulfonate Western Chemical Inc. MS-222  
Dissecting Scope Olympus SZ61 top SZX-ILLB2-100 base  
Confocal Microscope Olympus IX-81 with FV-1000 laser scanning confocal system  
TC-7 Tissue Culture Roller drum with 14 inch test tube wheel New Brunswick Scientific  TC-7  
Imaging Dishes MatTek Corporation P24G-1.0-10-F  
Pipette tips for loading needles Eppendorf 930001007  
Plate pouring grids Adaptive Science Tools TU-1
Heated Stage Bioptechs Inc. Delta T-5
Flat Spatula VWR Scientific 82027-486
Plastic Sieves Wares of Knutsford Online 12 cm
Parafilm VWR Scientific 52858-000
Vortex Genie VWR Scientific 14216-184
16 x 150 mm Culture tubes VWR Scientific 60825-435
Nanodrop Thermo Scientific ND 2000
Phenol Red VWR Scientific 97062-478
HCl VWR Scientific 87003-216
NaCl VWR Scientific BDH4534-500GP
KCl VWR Scientific BDH4532-500GP
MgSO4 VWR Scientific BDH0246-500GP
Ca(NO3)2 VWR Scientific BDH0226-500GP
HEPES VWR Scientific BDH4520-500GP
Morpholinos GeneTools, LLC

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Cite This Article
Brothers, K. M., Wheeler, R. T. Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae. J. Vis. Exp. (65), e4051, doi:10.3791/4051 (2012).

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