Non-invasiv Imaging af dissemineret candidiasis hos zebrafisk Larver
Summary
Den hastige udvikling, lille størrelse og gennemsigtighed i zebrafisk er enorme fordele for studiet af medfødte immunsystem kontrol af infektion<sup> 1-4</sup>. Her viser vi teknikker til at inficere zebrafisk larver ved hjælp af svampepatogen<em> Candida albicans</em> Ved mikroinjektion, for nylig metode, der anvendes til at indblande phagocyt NADPH oxidase aktivitet i kontrol af svampe dimorfi<sup> 5</sup>.
Abstract
Dissemineret candidiasis forårsaget af patogenet Candida albicans er en klinisk vigtigt problem indlagte individer og er forbundet med en 30 til 40% kan tilskrives mortalitet 6. Systemisk candidiasis normalt styres af medfødte immunitet, og individer med genetiske defekter i medfødte immunsystem cellekomponenter, såsom fagocyt NADPH-oxidase er mere modtagelige for candidemia 7-9. Meget lidt er kendt om dynamikken i C. albicans interaktion med medfødte immunsystem-celler in vivo. omfattende in vitro-undersøgelser har fastslået, at uden for værten C. albicans spirer indersiden af makrofager og hurtigt ødelægges af neutrofiler 10-14. In vitro-undersøgelser, men nyttige ikke rekapitulere komplekset in vivo-miljø, der omfatter tidsafhængige dynamik cytokinniveauer, ekstracellulære matrixproteiner vedhæftede filer og intercellulære kontakter 10, 15-18 </sup>. At undersøge bidraget af disse faktorer i værtspatogene interaktion, er det vigtigt at finde en modelorganisme at visualisere disse aspekter af infektionen ikke-invasivt på en levende intakt vært.
Zebrafisken larve giver en unik og alsidig vertebrat vært til undersøgelse af infektion. I de første 30 dage af udviklingen zebrafisk larver kun har medfødte immunforsvar 2, 19-21, forenkle undersøgelse af sygdomme, såsom dissemineret candidiasis, der er stærkt afhængige af medfødte immunitet. Den lille størrelse og gennemsigtighed i zebrafisk larver muliggøre scanning af infektion dynamik på det cellulære niveau for både vært og patogen. Transgene larver med fluorescerende medfødte immunsystem-celler kan anvendes til at identificere specifikke celletyper involveret i infektionen 22-24. Modificerede antisense-oligonukleotider (Morpholinos) kan anvendes til at vælte forskellige immun komponenter såsom fagocyt NADPH-oxidase og studere ændringer som reaktion på fungal infektion 5. Ud over de etiske og praktiske fordele ved at bruge en lille lavere hvirveldyr, tilbyder zebrafisk larverne unik mulighed for at billedet af slag mellem patogen og vært både intravitally og i farver.
Den Zebrafisken er blevet brugt til model-infektion for en række af de menneskelige sygdomsfremkaldende bakterier, og har været medvirkende til store fremskridt i vores forståelse af mycobakteriel infektion 3, 25. Men først for nylig har meget større patogener, såsom svampe blevet brugt til at inficere larve 5, 23, 26, og til dato har der ikke været en detaljeret visuel beskrivelse af infektionen metode. Her præsenterer vi vores teknikker til baghjerne ventrikel mikroinjektion af Prim 25 zebrafisk, herunder vores ændringer til tidligere protokoller. Vores resultater ved hjælp af larve zebrafisk model for svampeinfektion afvige fra in vitro undersøgelser og forstærke behovet for at undersøge vært-patogen vekselvirkningIndsatsen i komplekset miljø værten i stedet for en forenklet af petriskålen 5.
Protocol
Discussion
Den Zebrafisken mikroinjektion, der præsenteres her, adskiller sig fra Gutzman et al. 34 i dette her, vi viser injektion gennem otiske vesikel i baghjerne ventrikel 36 til 48 HPF larver. Metoden beskriver vi giver mulighed for konsekvent injektion af 10-15 gær i baghjerne ventrikel med reduceret vævsskader. Denne protokol giver en indledningsvis lokal infektion, der spredes i hele kroppen ved 24 HPI (figur 1) og resulterer i betydelig dødelighed / morbiditet 5. Den bag…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke laboratorium Dr. Carol Kim for mikroinjektion uddannelse, Clarissa Henry for at få råd om fremskyndelse embryo udvikling og anvendelse af udstyr, og Nathan Lawson for at bidrage fli1: EGFP fisk. Vi takker medlemmerne af Wheeler laboratoriet og Shawn Vægge til kritisk læsning af manuskriptet. Vi vil også gerne takke Mark Nilan for fisk pleje og rådgivning, og Ryan Phennicie og Kristin Gabor for teknisk rådgivning om dette projekt. Dette arbejde blev finansieret af en MAFES forskning assistentopholdet til K. Brothers, en MAFES Hatch tilskud E08913-08, og en NIH NCRR pris P20RR016463 til R. Wheeler.
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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