Name: non-invasive imaging of the innate immune response in a zebrafish larval model of streptococcus iniae infection
Uploaded: 2015-04-21T14:00:00
Description: Watch this Scientific Journal Video about Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection at JoVE.com

Forfatterne vil gerne takke lab medlemmer for zebrafisk pleje og vedligeholdelse. Dette arbejde blev støttet af National Institutes of Health, National Research Service Award A155397 til EA Harvie og NIH R01GM074827 til Anna Huttenlocher.

Voksne og embryonale zebrafisk blev opretholdt i overensstemmelse med University of Wisconsin-Madison Research Animal Resources Center. 1. Forberedelse Mikroinjektion Needles <ol><li> Forbered tynd væg glaskapillar kanyler (1,0 OD / 0,75 ID) ved hjælp af en mikropipette aftrækker enhed med følgende indstillinger: lufttryk 200, varme 502, træk 90, hastighed 80, tid 70, sendetid i starten af ​​pull 5, sendetid i slutningen af ​​pull 5. </li><li> Med fin pincet brække spidsen af...

<ol>
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Immun. 77 (11), 5190-5201 (2009).</li><li>Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: A Practical Approach. , (2002).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-mediated+experimental+metastasis+is+enhanced+by+VEGFR+inhibition+in+a+zebrafish+xenograft+model.">Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model.</a> J. Pathol. 227 (4), 431-445 (2012).</li><li>Haldi, M., Ton, C., Seng, W. 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Infektionen fremgangsmåden anvendt her er nyttig til undersøgelse af værtens immunrespons på en indledningsvis lokaliseret infektion i 2-3 DPF embryoner og larver. Fokus af en inflammatorisk stimulus, såsom infektion, i et lukket hulrum, såsom otisk vesikel muliggør studiet af neutrofile og makrofag kemotaxi og fagocytose. En advarsel injicere bakterier i otisk vesikel er, at evnen af ​​neutrofiler til effektivt fagocytere bakterier i væskefyldte hulrum kan være afhængig af den særlige mikrobe. Selv E...

Streptococcus iniae er en stor akvatisk patogen, der er i stand til at forårsage systemisk sygdom i både fisk og mennesker 1. Mens S. iniae er ansvarlig for store tab i akvakulturerhvervet, er det også en potentiel zoonotisk patogen, der kan forårsage sygdom hos immunkompromitterede menneskelige værter med kliniske patologier ligner dem, der forårsages af andre streptokok humane patogener. På grund af sine ligheder med humane patogener, er det vigtigt at studere S. iniae sygdom patogenese i forbindelse med en naturlig vært. En voksen zebrafisk model af S. iniae infektion afslørede robust infiltration af værten leukocytter til det lokaliserede sted for infektion samt en hurtig tid til vært død, en tid for kort involvere det adaptive immunsystem 7. For at få en grundig se på svar på S. medfødte immunforsvar iniae infektion in vivo, er det nødvendigt at anvende en model, som er mere modtagelig for non-invasiv direkte billedvisning. Larve zebrafisk har en række fordele, der gør det til et mere attraktivt hvirveldyr model til at studere vært-patogen interaktioner. Zebrafisk er relativt billige og nemme at bruge og vedligeholde i forhold til pattedyr modeller. Adaptive immunitet er ikke funktionelt modne indtil 4-6 uger efter befrugtningen, men larver har en højt konserveret hvirveldyr medfødte immunsystem med komplement, Toll-lignende receptorer, cytokiner og neutrofiler og makrofager med antimikrobielle egenskaber, herunder fagocytose og respiratorisk burst 2-6, 8-11. Derudover genetiske medgørlighed og optisk transparens af embryonale og larvestadier udvikling tillader generering af stabile transgene linjer med fluorescensmærkede immunceller gør det muligt at undersøge værtspatogene interaktioner i realtid in vivo. Frembringelsen af ​​disse transgene linjer ved hjælp af en photoconvertible protein, såsom Dendra2 muliggør sporing af individuelle værtscelle oprindelse og skæbne i løbet af infektion 12. Ved udvikling af en zebrafisk larver infektion model, vil det valgte sted for mikroinjektion afgøre, om en infektion er oprindeligt lokaliseret eller systemisk. Systemiske blod infektioner i halevenen eller Duct af Cuvier er mest almindeligt anvendte til at studere mikrobielle patogener i zebrafisk og er nyttige til at studere samspillet mellem vært og mikrobielle celler, cytokinresponser, og forskelle i virulens mellem patogene stammer. For langsommere voksende mikroorganismer, kan tidlig injektion i blommesækken af et embryon på 16-1,000 cellestadiet anvendes til at frembringe en systemisk infektion 13,14, med den optimale udviklingsstadiet for mikroinjektion af en langsomt voksende mikroorganisme fundet at være mellem den 16-128 cellestadiet 15. Men blommesækken injektioner af mange mikrober på senere stadier af vært udvikling tendens til at være dødelig til than vært på grund af den næringsrige miljø for mikrobe og manglende infiltrerende leukocytter 16-18. En lokaliseret infektion normalt resulterer i rettet vandring af leukocytter i retning af infektionsstedet, der let kan kvantificeres med non-invasiv billeddannelse. Denne type infektion kan tillade dissektion af de mekanismer, der formidler leukocytmigration samt undersøgelse af forskellige vandrende og fagocytiske kapacitet af forskellige leukocytpopulationer. Lokaliserede infektioner er også nyttige i forbindelse med behandlingen forskelle i virulens mellem bakteriestammer samt studere mikrobe invasion mekanismer siden fysiske barrierer vært skal krydses for en lokaliseret infektion bliver systemisk. Zebrafisk typisk rejses ved temperaturer på 25-31 ° C 19, men de kan også holdes ved temperaturer så høje som 34-35 ° C til undersøgelser af invasiv visse humane patogener med strenge temperaturkravfor virulens 20, 21. Mange forskellige steder er blevet anvendt til at generere en indledningsvis lokaliseret bakteriel infektion, herunder baghjerne ventrikel 22, dorsale hale muskel 18 perikardiehulrummet 23 og otisk vesikel (øre) 5, 16, 24. Det har imidlertid vist sig, at injektion af bakterier i halen muskel kan forårsage vævsskade og inflammation uafhængig af de bakterier, der kan forvrænge resultaterne, når de undersøger leukocyt reaktion 13. Selv mindre skade er forbundet med injektion i baghjerne og selv om det er i første omgang uden leukocytter hos unge embryoner, baghjerne ventrikel støt får mere immunceller over tid, da mikroglia tage ophold. Baghjerne ventrikel er også en mere vanskelig placering af billedet. Den otisk vesikel er et lukket hulrum uden direkte adgang til vaskulaturen 25, 26. Det er normalt uden leukocytes, men leukocytter kan rekrutteres til otisk vesikel som reaktion på inflammatoriske stimuli, såsom infektion. Det er også et foretrukket sted for mikroinjektion af bakterier i zebrafisk alderen 2-3 dage efter befrugtning (DPF) på grund af den lethed, billedbehandling og visualisering af injektionen. Derfor valgte vi otisk vesikel som vores hjemmeside af lokaliseret bakteriel infektion.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1.7 ml eppendorfs</td>
			<td>MidSci</td>
			<td>AVSS1700</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml falcon tube</td>
			<td>BD Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 1/2 in. needle</td>
			<td>BD Biosciences</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning Incorporated</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD Biosciences</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA agar</td>
			<td>Dot Scientific, Inc</td>
			<td>7126A</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-7m</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>MDS</td>
			<td>2292</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine tweezers</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel comb</td>
			<td>VWR</td>
			<td>27372-482</td>
			<td>4.2 mm width, 1.5 mm thick</td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td></td>
			<td></td>
			<td>Custom made by drilling a 16&#8211;18 mm hole in the center of a 35-mm tissue culture dish bottom and placing a 22-mm round #1 coverslip in the hole and sealing with a thin layer of Norland Optical Adhesive 68 cured by UV light.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>High melt agarose</td>
			<td>Denville Scientific, Inc.</td>
			<td>CA3510-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Fisher Scientific</td>
			<td>H325</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>with FV-1000 system</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melt agarose</td>
			<td>Fisher</td>
			<td>BP165-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Tritech (Narishige)</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection system</td>
			<td>Parker</td>
			<td>Picospritzer III</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipet tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Tritech (Narishige)</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument Company</td>
			<td>Flaming/Brown P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotmeter</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldheyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td>100 mm x 15 mm</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma Aldrich</td>
			<td>P-4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Ricca Chemoical Company</td>
			<td>572516</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>BP665-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>P-6310</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Roche</td>
			<td>165921</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease peptone</td>
			<td>Fluka Biochemika</td>
			<td>29185</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cell culture dish</td>
			<td>Corning Incorporated</td>
			<td>430165</td>
			<td>35 mm x 10 mm</td>
		</tr>
		<tr>
			<td>Sudan Black</td>
			<td>Sigma Aldrich</td>
			<td>S2380</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall glass capillary injection needles</td>
			<td>World Precision Instruments, Inc.</td>
			<td>TW100-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Todd Hewitt</td>
			<td>Sigma Aldrich/Fluka Analytical</td>
			<td>T1438</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (ethyl 3-aminobenzoate)</td>
			<td>Argent Chemical Laboratory/Finquel</td>
			<td>C-FINQ-UE-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fluka Biochemika</td>
			<td>92144</td>
			<td></td>
		</tr>
	</tbody>
</table>

non-invasive imaging of the innate immune response in a zebrafish larval model of streptococcus iniae infection

Den akvatiske patogen, Streptococcus iniae, er ansvarlig for mere end 100 millioner dollars i årlige tab for akvakulturerhvervet og er i stand til at forårsage systemisk sygdom i både fisk og mennesker. En bedre forståelse af S. iniae sygdom patogenese kræver en passende model system. Den genetiske sporbarhed og optisk transparens af de tidlige udviklingsstadier af zebrafisk muliggør generering og ikke-invasiv billeddannelse af transgene linjer med fluorescerende mærkede immunceller. Det adaptive immunsystem er ikke fuldt funktionelt før adskillige uger efter befrugtning, men zebrafisk larver har et konserveret hvirveldyr medfødte immunsystem med både neutrofiler og makrofager. Således frembringelsen af en larve infektion model tillader studiet af specifikke bidrag medfødte immunitet kontrollere S. iniae infektion. Webstedet for mikroinjektion vil afgøre, om en infektion ersystemisk eller oprindeligt lokaliseret. Her præsenterer vi vores protokoller for otisk vesikel injektion af zebrafisk alderen 2-3 dage efter befrugtningen og vores teknikker til fluorescerende konfokal billeddannelse af infektion. En lokaliseret infektion site giver observation af første mikrobe invasion, rekruttering af værtsceller og formidling af infektion. Vores resultater ved hjælp af zebrafisk larver model S. iniae infektion indikerer, at zebrafisk kan anvendes til at undersøge de forskellige bidrag værten neutrofiler og makrofager i lokaliserede bakterieinfektioner. Desuden beskriver vi, hvordan photolabeling af immunceller kan bruges til at spore individuel værtscelle skæbne under infektion.

Mikroinjektion af S. iniae til otisk vesikel (figur 1 og figur 2) resulterer i en indledningsvis lokaliseret vært respons. Når det injiceres korrekt, bør bakterierne kun ses i otisk vesikel og ikke i det omgivende væv eller blod. Dette kan visualiseres under mikroinjektion anvendelse af phenol rødt farvestof (figur 1A). Alternativt, hvis mærkede bakterier injiceres, en hurtig scanning af inficerede larver straks efter injektion kan bekræfte bakterierne e...

Watch this Scientific Journal Video about Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection at JoVE.com

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

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Ikke-invasiv billeddannelse af Innate immunrespons i en zebrafisk Larve Model af<em&gt; Streptococcus iniae</em&gt; Infektion

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Research

JoVE Journal

Immunology and Infection

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Non-invasiv Imaging af dissemineret candidiasis hos zebrafisk Larver

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

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Ikke-invasiv optisk billeddannelse af lymfatisk vaskulatur på en mus

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

Kvantificering af den Respiratory burstrespons som en indikator for medfødte immunforsvar Sundhed i zebrafisk

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

Dissekere medfødte immunforsvar signalering i Viral Evasion af cytokinproduktion

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Brug af RNA-interferens Undersøge Innate immunresponset i musemakrofager

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-invasive Model of Neuropathogenic<em&gt; Escherichia coli</em&gt; Infektion i Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In vitro analyse af MyD88-medieret Cellular immunrespons til West Nile Virus mutantstamme Infektion

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...