Неинвазивная визуализация диссеминированный кандидоз в Личинки данио рерио
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
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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