Name: Forfatter Spotlight: Etablering af kimfri zebrafisk til mikrobiota-værtsforholdsundersøgelser
Uploaded: 2024-04-12T13:50:00
Description: Watch this Scientific Journal Video about Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies at JoVE.com

Vi takker oprigtigt støtten fra Chongqing Medical University Talent Project (nr. R4014 til DSP og R4020 til PPJ), National Natural Science Foundation of China (NSFC, nr. 32200386 til PPJ), Chongqing Postdoctoral Innovation Mentor Studio (X7928 DSP) og Program for Kina-Sri Lanka Joint Center for Water Technology Research and Demonstration af Chinese Academy of Sciences (CAS) / Kina-Sri Lanka Joint Center for Education and Research af CAS.

Fiskeforsøgene blev udført i overensstemmelse med retningslinjerne fra Animal Care and Use Committee of Chongqing og Institutional Animal Care and Use Committee of Chongqing Medical University, Kina, samt standarderne for forsøgsdyr udstedt af State Bureau of Quality and Technical Supervision (godkendelses-id: GB14922-2001 til GBT14927-2001). Zebrafisk (Danio rerio, vildtype, AB-stamme) blev hentet fra Institute of Hydrobiology, Chinese Academy of Sciences og opretholdt i laboratoriet efter tidligere rapporte...

Optimering af GF Zebrafish-modeller: Generering, vedligeholdelse og mikrobielle undersøgelser

Kritiske trin inden for protokollerne for GF fisk og GF madlavning Under genereringen af GF-fiskemodeller var flere kritiske trin involveret, herunder fremstilling af sterile materialer, sterilisering af embryoner, daglig fornyelse af GZM, indsamling af forskellige prøver og steril undersøgelse af hver prøve ved hjælp af flere metoder. Blandt disse trin er den indledende behandling af embryoner grundlæggende og afgørende for GF-modellernes succes. Kontrollerende midler, deres koncentrationer og ...

Mikrobiota (dvs. arkæer, bakterier, eukarya og vira) spiller afgørende roller for at opretholde værtssundhed og bidrage til udviklingen af forskellige sygdomme ved at påvirke fysiologiske og patologiske processer gennem symbiotiske interaktioner inden for tarmbarrieren, epiteloverfladen og mucinfunktioner hos individer 1,2,3. Sammensætningen af mikrobiota på tværs af forskellige livsstadier, fra barndom til juvenilitet, voksenalder og aldring, samt dens tilstedeværelse på forskellige steder såsom nares, oral, hud og tarmsteder, er dynamisk formet af forskellige levesteder og miljøer4. Den intestinale mikrobiota i organismer er involveret i næringsabsorption, immunrespons, patogeninvasion, metabolisk regulering osv. 5,6. Undersøgelser af patienter har vist, at forstyrrelser i tarmmikrobiota er relateret til menneskelig fedme, søvnforstyrrelser, depression, inflammatorisk tarmsygdom (IBD), neurodegenerative sygdomme (Parkinsons, Alzheimers), aldring og forskellige kræftformer 7,8,9. Desuden involverer interaktive veje mellem tarmmikrobiota og værter inflammatoriske faktorer, neurotransmittere, metabolitter, tarmbarriere og oxidativt stress, som observeret i tidligere forskning ved hjælp af mus og fiskemodeller10,11.
For nylig er flere bakterierelaterede tilgange eller terapier, herunder potentielle probiotika og fækal mikrobiotatransplantation (FMT), blevet undersøgt for disse lidelser i kliniske og dyremodeller. Disse udforskninger er baseret på opdagelser relateret til mikrobiota-tarm-hjerne / lever / nyreaksen, mikrobiotaafledte produkter og ændret receptoraktivitet12,13. Imidlertid er udviklingen, forskellige funktioner og mekanismer i mikrobiota-værtssystemet stadig ufuldstændigt forstået og identificeret på grund af kompleksiteten af det mikrobielle samfund og udfordringen med at generere kraftfulde menneskelignende sygdomsmodeller.
For at løse disse problemer blev kimfrie (GF) dyremodeller hurtigt foreslået i midten af det 19. århundrede og primært udviklet i løbetaf det 20. århundrede. Efterfølgende forbedringer, herunder antibiotikabehandlede og gnotobiotiske modeller, sammen med fremskridt inden for mikrobiel detektion og observationsteknologier, perfektionerede yderligere disse modeller 14,15,16. GF-dyr, skabt ved at slette deres egen baggrund og undgå miljømikrober, tilbyder en fremragende strategi til at udforske interaktionerne mellem mikroorganismer og deres værter17. Gennem anvendelse af dyremodeller og raffinerede protokoller har forskere med succes replikeret lignende mikrobielle sammensætninger, der findes hos patienter i GF-mus og fisk. Derudover giver andre GF-dyremodeller, såsom hunde, kyllinger og svin, forskellige muligheder som forskningsemner 18,19,20,21. Denne tilgang har muliggjort undersøgelser af de potentielle terapeutiske virkninger af kommensale mikrobiomer på forskellige sygdomme, herunder kræftimmunterapi hos mennesker16,18. GF-modeller giver mere præcis indsigt i karakteristika og mekanismer for specifik bakteriel kolonisering, migration, multiplikation og interaktion inden for værter. Dette giver afgørende ny indsigt i forekomsten og udviklingen af mikrobiota-relaterede sygdomme22,23. Historien om etablering og anvendelse af GF-zebrafisk i mikrobiel forskning har udviklet sig fra rapporterne fra Rawls et al. i 2004 og Bates et al. i 2006 til Melancon et al.'s protokol i 2017 16,24,25. Imidlertid er gennemførligheden af voksne eller avls GF-modeller stadig en langvarig proces ledsaget af variabel levetid, succesrater og sundhedsmæssige udfordringer.
Blandt forskellige dyremodeller skiller zebrafisk (Danio rerio) sig ud som et kritisk værktøj til både grundlæggende og biomedicinsk forskning på grund af dets fordelagtige lighed med menneskelige organer og genomik, kort udviklingscyklus, høj frugtbarhed og gennemsigtige embryoner19,26. Zebrafisk, der tjener som pålidelige humane sygdomsmodeller, tilbyder en visuel repræsentation af fysiologiske og patologiske processer in vivo, hvilket giver indsigt i de attraktive træk ved værtsmikrobeinteraktioner. Især udviser zebrafisk forskellige cellelinjer, hvilket muliggør billeddannelse af tarmfysiologi, mikrobiel dynamik, gonader og reproduktiv udvikling, modning af værtsimmunsystemet, adfærd og metabolisme27. Zebrafiskembryoner udvikler sig inden for beskyttende korioner indtil udklækning og bliver larver 3 dage efter befrugtning (dpf). De jager aktivt efter mad ved 5 dpf og når seksuel modenhed omkring 3 måneder efter befrugtning (mpf)28. Den første vellykkede kimfri (GF) zebrafisk, rapporteret af Rawls et al.24, viste, at larver fodret med autoklaveret foder efter æggeblommeabsorption udviste vævsnekrose fra 8 dpf og total død ved 20 dpf. Dette indikerede virkningerne af kost eller vigtigheden af at overveje eksogen næringsstofforsyning i forsøg, der involverede langsigtede (>7 dpf) GF-fisk29. Efterfølgende undersøgelser forbedrede generationsprotokollen for GF-fisk ved at anvende steril mad og metoder perfektioneret i forskellige fiskemodeller16.
Imidlertid har det meste forskning på GF zebrafiskmodeller fokuseret på tidlige livsstadier, der involverer bakteriel infektion ved 5 dpf i 24 timer til 48 timer, med prøver indsamlet før 7 dpf ved afslutningen af forsøgene 25,30,31. Det er almindeligt anerkendt, at mikrobiota i organismer, herunder mennesker og zebrafisk, koloniseres i begyndelsen af livet og formes under vækst og udvikling. Sammensætningen forbliver stabil i voksne stadier, hvor mikrobiotas roller i værten er afgørende gennem hele livet, især i aldring, neurodegenerativ, metabolisk relateret fedme og tarmsygdomsaspekter3. Således kan perspektiver fra GF-dyr med længere overlevelse give indsigt i mekanismerne for mikrobielle roller i værtsorganudvikling og funktioner i betragtning af fiskelarvernes umodne immun- og reproduktionssystemer tidligt i livet. Mens bakteriestammer i zebrafiskens tarme er blevet isoleret og identificeret i tidligere undersøgelser, hvilket giver potentialet til at inficere GF-dyremodeller til at vælge probiotika eller forskningsbakterielle funktioner i værten19,25, har generering og anvendelse af GF-fiskemodeller primært været begrænset til tidlige livsstadier. Denne begrænsning, der tilskrives den komplekse produktionsproces, høje vedligeholdelsesomkostninger og tilknyttede problemer med mad og immunitet, hindrer forskningsindsats, der sigter mod at undersøge de udviklingsmæssige og kroniske virkninger af mikrobiota i værten.
Overlevelsesraten, adfærd, vækst, modning og generel sundhed hos fisk, især i kimfrie (GF) modeller, påvirkes signifikant af fodringspraksis, der omfatter ernæringsindtag og absorption i den åbne periode fra tidlige larver til unge32,33. En af udfordringerne i GF-fiskeopdræt er imidlertid manglen på egnede sterile kostvaner, hvilket begrænser effektiviteten af ernæringsmæssig støtte til opretholdelse af larvernes vækst og overlevelse. Løsning af dette problem er afgørende for at genoprette GF-fiskens liv i betragtning af deres udviklingsforsvarsmekanismer og svage fordøjelsesevner på grund af fraværet af et tarmmikrobiom. Med hensyn til mad fremstår levende artemia (Artemia sp.) som den mest egnede diæt til mundåbne larver til unge fisk. Det er blevet observeret, at fisk, der fodres med levende artemia, udviser højere vækst- og overlevelsesrater sammenlignet med dem, der fodres med kogt æggeblomme eller andre naturlige og syntetiske lokkemad34. Mens tidlige livsmodeller af GF-fisk kan overleve med æggeblommestøtte, og GF-larvermodeller kan opretholdes med steril fodring, er det stadig udfordrende at generere langsigtede modeller fra larver til unge og nå seksuel modenhed. Derudover er flage- eller pulverfoder begrænset af ulige ernæringssammensætning og kan påvirke vandkvaliteten. I modsætning hertil har levende Artemia fordele som overlevelse i både salt og ferskvand, lille størrelse egnet til larver til voksne, let batching og højere rugekvalitet35. Med udgangspunkt i tidligere metoder 16,24,30 har vi forenklet den komplekse behandlingsproces og adresseret kostudfordringen ved at etablere let inkuberet GF levende Artemia sp. som steril mad i længere varighed end GF-fisk i det tidlige liv.
Denne undersøgelse præsenterer en optimeret protokol, der dækker (1) generation, (2) vedligeholdelse, (3) identifikation af steril hastighed og (4) vedligeholdelse og fodring for at sikre væksten af kimfri (GF) zebrafisk fra embryoner til larver og unge stadier. Resultaterne giver foreløbige beviser for udklækning, overlevelse, vækst og sterilitet af GF zebrafisk sammen med vigtige indekser for GF Artemia sp. som steril mad. De detaljerede trin i modelgenerering og tilberedning af sterile levende fødevarer giver afgørende teknisk støtte til konstruktion og anvendelse af langsigtede GF-fiskemodeller samt GF Artemia sp. i mikrobiota-værtsinteraktionsforskning. Protokollen omhandler bakteriel isolering, identifikation og infektion på GF-fiskemodeller, der skitserer metoder til bakteriel fluorescensmærkning og observerer deres kolonisering i fisketarme under et mikroskop. GF-fisk, gnotobiotiske fisk med bakteriel infektion eller overførte humane mikrobiotamodeller vil gennemgå forskellige påvisninger for at belyse deres funktioner og virkninger på værtsimmunitet, fordøjelse, adfærd, transkriptomisk regulering og metaboliske aspekter. På lang sigt kan denne protokol udvides til forskellige vildtypefiskearter, såsom marine medaka, og potentielt til andre udvalgte transgene zebrafisklinjer, der er korreleret med specifikke væv eller sygdomme.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AB-GZM</td>
			<td>Amphotericin:Solarbio;&#160; kanamycin:Solarbio; Ampicillin:Solarbio.</td>
			<td>Amphotericin:CAS:1397-89-3; 
			kanamycin:CAS: 25380-94-0; Ampicillin:CAS: 69-52-313.</td>
			<td>49.6 mL GZM, 50 &#181;L amphotericin stock solution (250 &#181;g/mL), 25 &#181;L kanamycin stock solution (10 mg/mL), and 250 &#181;L ampicillin stock solution (20 mg/mL).</td>
		</tr>
		<tr>
			<td>1.5 mL, 15 mL, 50 mL EP tubes</td>
			<td>biosharp</td>
			<td>BS-15-M</td>
			<td>To collect samples, and hold agents</td>
		</tr>
		<tr>
			<td>2.4 g/L NaClO</td>
			<td>XILONG SCIENTIFIC Co., Ltd.</td>
			<td>CAS: 7681-52-9</td>
			<td>Diluted with 8% sodium hypochlorite aqueous solution.</td>
		</tr>
		<tr>
			<td>6-well plates, 24-, 48- well plates</td>
			<td>LABSELECT&#160;</td>
			<td>11112</td>
			<td>To culture fish</td>
		</tr>
		<tr>
			<td>Aeronomas</td>
			<td>NCBI database</td>
			<td>No.MK178499</td>
			<td>2019-JPP-ESN</td>
		</tr>
		<tr>
			<td>Anaerobic TSA plates</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp; 
			agar powder:BioFroxx.</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112; 
			agar powder:9002-18-0.</td>
			<td>The TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, and 6 g agar powder under the anaerobic system.</td>
		</tr>
		<tr>
			<td>Anaerobic work station</td>
			<td>GENE SCIENCE</td>
			<td>E200G</td>
			<td>Bacterial isolation, sterile testing</td>
		</tr>
		<tr>
			<td>Analysis</td>
			<td>GraphPad Prism 5</td>
			<td>v6.07</td>
			<td>To analysis the data</td>
		</tr>
		<tr>
			<td>API 20 E kits</td>
			<td>&#160;BioMerieux SA, France</td>
			<td>No.1005915090</td>
			<td>Ref 20100 Kits to detect bacterial metabolism</td>
		</tr>
		<tr>
			<td>Artemia (Brine shrimp)</td>
			<td>Shangjia Aquarium Co., Ltd.</td>
			<td>Aquamaster brand</td>
			<td>Artemia cysts, and brine shrimp eggs&#160;</td>
		</tr>
		<tr>
			<td>Auto cycle system for fish culture</td>
			<td>Ningbo Hairui Technology Co., Ltd</td>
			<td>No Cat</td>
			<td>Maintain the fish</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Zeal Way</td>
			<td>G154DWS</td>
			<td>Prepare the materials</td>
		</tr>
		<tr>
			<td>BHI Aerobic</td>
			<td>Coolaber</td>
			<td>Cat#PM0640</td>
			<td>BHI medium was prepared, wherein 100 mL medium included 3.7 g BHI powder.</td>
		</tr>
		<tr>
			<td>BHI Anaerobic</td>
			<td>Coolaber</td>
			<td>Cat#PM0640</td>
			<td>BHI medium was prepared and divided into anaerobic tubes under the anaerobic system.</td>
		</tr>
		<tr>
			<td>Biochemical incubator</td>
			<td>LongYue Co., Ltd</td>
			<td>SPX</td>
			<td>For fish and plates</td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Haier</td>
			<td>HR40-IIA2</td>
			<td>Sterile treatment and testing</td>
		</tr>
		<tr>
			<td>Bleaching agent of 0.02 g/L NaClO</td>
			<td>XILONG SCIENTIFIC Co., Ltd.</td>
			<td>CAS: 7681-52-9</td>
			<td>Working solution with sodium hypochlorite (NaClO) concentration: Diluted with 8% sodium hypochlorite aqueous solution or 166.6 uL 6% sodium hypochlorite with 500 mL distilled water.</td>
		</tr>
		<tr>
			<td>Blood plates</td>
			<td>sheep blood:Solarbio</td>
			<td>Cat. NO. TX0030</td>
			<td>Sterile-defibrinated sheep blood was added into TSA to prepare 5% blood plates.</td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td>To culture fish</td>
		</tr>
		<tr>
			<td>CM-Dil dyes</td>
			<td>Molecular Probes</td>
			<td>Cat#C7000&#160;&#160;</td>
			<td>To label the bacteria</td>
		</tr>
		<tr>
			<td>Constant temperature shaking incubator</td>
			<td>Peiving Co., Ltd</td>
			<td>HZQ-X100</td>
			<td>Bacterial culture</td>
		</tr>
		<tr>
			<td>Database</td>
			<td>NCBI</td>
			<td>Bacteria and Archaea database</td>
			<td>Link: Archaea FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Archaea/ 
			Bacteria FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Bacteria/</td>
		</tr>
		<tr>
			<td>Disposable Pasteur pipette</td>
			<td>biosharp</td>
			<td>bs-xh-03l</td>
			<td>Used to change water, and transfer eggs</td>
		</tr>
		<tr>
			<td>Disposable petri dish</td>
			<td>biosharp</td>
			<td>BS-90-D</td>
			<td>To culture fish</td>
		</tr>
		<tr>
			<td>DNA kits</td>
			<td>Solaribio</td>
			<td>Cat#D1600</td>
			<td>Bacterial genomic DNA extraction kits&#160;</td>
		</tr>
		<tr>
			<td>Electric pipette</td>
			<td>SCILOGEX</td>
			<td>Levo me</td>
			<td>Change water</td>
		</tr>
		<tr>
			<td>Exiguobacterium</td>
			<td>NCBI database</td>
			<td>No.MK178504</td>
			<td>2019-JPP-ESN</td>
		</tr>
		<tr>
			<td>GZM</td>
			<td>Sea salt:LANDEBAO Co., Ltd.</td>
			<td>No Cat</td>
			<td>Composed of 1 L of water and 1.5 mL of sea salt solution (40 g/L), autoclaved. The content of sea salt in the GZM solution was 60 mg/L.</td>
		</tr>
		<tr>
			<td>Laboratory pure water system</td>
			<td>Hitech Co., Ltd</td>
			<td>Prima-S15</td>
			<td>Prepare the agents</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td>With fluorescent light to observe fish larvae</td>
		</tr>
		<tr>
			<td>PCR kits</td>
			<td>TIANGEN</td>
			<td>Cat#ET101</td>
			<td>Taq DNA Polymerase kit</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>LABSELECT&#160;</td>
			<td>sp-013-10</td>
			<td>Change water</td>
		</tr>
		<tr>
			<td>Povidone iodine (PVP-I)</td>
			<td>Aladdin</td>
			<td>Lot#H1217005</td>
			<td>Aqueous solution povidone iodine 0.4 g/L pure water.</td>
		</tr>
		<tr>
			<td>Timing converter</td>
			<td>PinYi Co., Ltd</td>
			<td>AL-06</td>
			<td>To regulate the light</td>
		</tr>
		<tr>
			<td>TSA plates</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp; 
			agar powder:BioFroxx.</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112; 
			agar powder:9002-18-0.</td>
			<td>TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, 6 g agar powder.</td>
		</tr>
		<tr>
			<td>TSB Aerobic</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp;</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112;</td>
			<td>TSB medium was prepared, wherein 400 mL medium included 6 g tryptone, 2 g soy peptone, and 2 g NaCl.</td>
		</tr>
		<tr>
			<td>TSB Anaerobic</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp;</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112;</td>
			<td>TSB medium was prepared and divided into the anaerobic tubes under the anaerobic system.</td>
		</tr>
		<tr>
			<td>Ultra-clean workbench</td>
			<td>Airtech</td>
			<td>SW-CJ-2FD</td>
			<td>Sterile treatment and testing</td>
		</tr>
		<tr>
			<td>Ultra-pure flow system for fish culture</td>
			<td>Marine Biological Equipment company</td>
			<td>No Cat</td>
			<td>Produce water for fish</td>
		</tr>
		<tr>
			<td>Vibrio</td>
			<td>NCBI database</td>
			<td>No.MK178501</td>
			<td>2019-JPP-ESN</td>
		</tr>
	</tbody>
</table>

generation, maintenance, and identification of germ-free zebrafish models from larvae to juvenile stages

Zebrafisk tjener som værdifulde modeller for forskning i vækst, immunitet og tarmmikrobiota på grund af deres genomiske ligheder med pattedyr, gennemsigtige embryoner udviklet i et relativt rent chorionmiljø og ekstremt hurtig udvikling af larver sammenlignet med gnavermodeller. Kimfri (GF) zebrafisk (Danio rerio) er afgørende for evaluering af forurenende toksicitet og etablering af menneskelignende sygdomsmodeller relateret til mikrobielle funktioner. I sammenligning med konventionelt opdrættede (CR) modeller (fisk i almindeligt husdyrhold) giver GF-zebrafisk mulighed for mere nøjagtig manipulation af værtsmikrobiotaen, hvilket hjælper med at bestemme årsagssammenhængen mellem mikroorganismer og værter. Derfor spiller de en afgørende rolle i at fremme vores forståelse af disse forhold. GF zebrafiskmodeller genereres og undersøges dog typisk i de tidlige livsstadier (fra embryoner til larver) på grund af begrænsninger i immunfunktion og næringsabsorption. Denne undersøgelse optimerer generering, vedligeholdelse og identifikation af tidlige GF zebrafiskmodeller uden fodring og med langvarig fodring ved hjælp af GF-mad (såsom Artemia sp., artemia). Gennem hele processen blev daglig prøveudtagning og kultur udført og identificeret gennem flere detektioner, herunder plader og 16S rRNA-sekventering. De aseptiske hastigheds-, overlevelses- og udviklingsindekser for GF-zebrafisk blev registreret for at sikre kvaliteten og kvantiteten af de genererede modeller. Det er vigtigt, at denne undersøgelse giver detaljer om bakteriel isolering og infektionsteknikker for GF-fisk, hvilket muliggør effektiv oprettelse af GF-fiskemodeller fra larver til ungdomsstadier med GF-fødevarestøtte. Ved at anvende disse procedurer i biomedicinsk forskning kan forskere bedre forstå forholdet mellem tarmbakteriefunktioner og værtssundhed.

The microbiota (i.e., Archaea, Bacteria, Eukarya, and viruses) play crucial roles in maintaining host health and contributing to the development of various diseases by influencing physiological and pathological processes through symbiotic interactions within the intestinal barrier, epithelial surface, and mucin functions in individuals1,2,3. The composition of the microbiota across different life stages, from infancy to juvenility, adulthood, and aging, as well as its presence in various locations such as nares, oral, skin, and gut sites, is dynamically shaped by diverse habitats and environments4. The intestinal microbiota in organisms is involved in nutrient absorption, immune response, pathogen invasion, metabolic regulation, etc5,6. Studies on patients have demonstrated that disruptions in gut microbiota are related to human obesity, sleep disorders, depression, inflammatory bowel disease (IBD), neurodegenerative diseases (Parkinson&#39;s, Alzheimer&#39;s), aging, and various cancers7,8,9. Furthermore, interactive pathways between gut microbiota and hosts involve inflammatory factors, neurotransmitters, metabolites, intestinal barrier, and oxidative stress, as observed in previous research using mice and fish models10,11.
Recently, multiple bacteria-related approaches or therapies, including potential probiotics and fecal microbiota transplantation (FMT), have been explored for these disorders in clinical and animal models. These explorations are based on discoveries related to the microbiota-gut-brain/liver/kidney axis, microbiota-derived products, and altered receptor activity12,13. However, the development, various functions, and mechanisms of the microbiota-host system are still incompletely understood and identified due to the complexity of the microbial community and the challenge of generating powerful human-like disease models.
To address these issues, germ-free (GF) animal models were urgently proposed in the mid-19th century and primarily developed during the 20th century. Subsequent refinements, including antibiotic-treated and gnotobiotic models, along with advancements in microbial detection and observation technologies, further perfected these models14,15,16. GF animals, created by erasing their own background and avoiding environmental microbes, offer an excellent strategy for exploring the interactions between microorganisms and their hosts17. Through the application of animal models and refined protocols, researchers have successfully replicated similar microbial compositions found in patients in GF mice and fish. Additionally, other GF animal models, such as dogs, chickens, and pigs, provide diverse options as research subjects18,19,20,21. This approach has enabled investigations into the potential therapeutic effects of commensal microbiomes on various diseases, including cancer immunotherapy in humans16,18. GF models offer more accurate insights into the characteristics and mechanisms of specific bacterial colonization, migration, multiplication, and interaction within hosts. This provides crucial novel insights into the occurrence and development of microbiota-related diseases22,23. The history of establishing and applying GF zebrafish in microbial research has evolved from the reports of Rawls et al. in 2004 and Bates et al. in 2006 to Melancon et al.&#39;s protocol in 201716,24,25. However, the feasibility of adult or breeding GF models is still a prolonged process, accompanied by variable longevity, success rates, and health challenges.
Among various animal models, zebrafish (Danio rerio) stands out as a critical tool for both basic and biomedical research due to its advantageous similarity to human organs and genomics, short developmental cycle, high fecundity, and transparent embryos19,26. Zebrafish, serving as reliable human disease models, offer a visual representation of physiological and pathological processes in vivo, providing insights into the attractive features of host-microbe interactions. Notably, zebrafish exhibit distinct cell lineages, allowing imaging of intestinal physiology, microbial dynamics, gonads and reproductive development, maturation of the host immune system, behavior, and metabolism27. Zebrafish embryos develop within protective chorions until hatching, becoming larvae at 3 days post-fertilization (dpf). They actively hunt for food at 5 dpf and reach sexual maturity around 3 months post-fertilization (mpf)28. The first successful germ-free (GF) zebrafish, reported by Rawls et al.24, showed that larvae fed with autoclaved feed after yolk absorption exhibited tissue necrosis from 8 dpf and total death at 20 dpf. This indicated the effects of diet or the importance of considering exogenous nutrient supply in experiments involving long-term (&#62;7 dpf) GF fish29. Subsequent studies improved the generation protocol of GF fish, employing sterile food and methods perfected in different fish models16.
However, most research on GF zebrafish models has focused on early life stages, involving bacterial infection at 5 dpf for 24 h to 48 h, with samples collected before 7 dpf at the conclusion of the experiments25,30,31. It&#39;s widely acknowledged that the microbiota in organisms, including humans and zebrafish, is colonized at the beginning of life and shaped during growth and development. The composition remains stable at adult stages, with the roles of microbiota in the host being crucial throughout life, especially in aging, neurodegenerative, metabolic-related obesity, and intestinal disease aspects3. Thus, perspectives from GF animals with longer survival can provide insights into the mechanisms of microbial roles in host organ development and functions, considering the immature immune and reproductive systems of fish larvae in early life. While bacterial strains in zebrafish intestines have been isolated and identified in previous studies, offering the potential for infecting GF animal models to select probiotics or research bacterial functions in the host19,25, the generation and application of GF fish models have primarily been restricted to early life stages. This limitation, attributed to the complex production process, high maintenance costs, and associated issues with food and immunity, hinders research efforts aimed at investigating the developmental and chronic effects of microbiota in the host.
The survival rate, behavior, growth, maturation, and overall health of fish, especially in germ-free (GF) models, are significantly influenced by feeding practices, encompassing nutrition intake and absorption during the mouth-open period from early larvae to juveniles32,33. However, one of the challenges in GF fish husbandry is the scarcity of suitable sterile diets, limiting the effectiveness of nutritional support for sustaining the growth and survival of larvae. Resolving this issue is crucial to restoring the life of GF fish, considering their developmental defense mechanisms and weak digestion abilities due to the absence of an intestinal microbiome. In terms of food, live brine shrimp (Artemia sp.) emerges as the most suitable diet for mouth-open larvae to juvenile fish. It has been observed that fish fed with live brine shrimp exhibit higher growth and survival rates compared to those fed with cooked egg yolk or other natural and synthetic baits34. While early life models of GF fish can survive with yolk support and GF larvae models can be maintained with sterile feeding, generating long-term models from larvae to juveniles and reaching sexual maturity remains challenging. Additionally, flake or powder food is limited by unequal nutritional composition and can impact water quality. In contrast, live Artemia has advantages such as survival in both salt and freshwater, small size suitable for larvae to adults, ease of batching, and higher hatching quality35. Building upon previous methods16,24,30, we have simplified the complex treatment process and addressed the diet challenge by establishing easily incubated GF live Artemia sp. as sterile food for longer durations than early-life GF fish.
This study presents an optimized protocol covering (1) generation, (2) maintenance, (3) identification of sterile rate, and (4) maintenance and feeding to ensure the growth of germ-free (GF) zebrafish from embryos to larvae and juvenile stages. The results offer preliminary evidence on the hatching, survival, growth, and sterility of GF zebrafish, along with essential indices for GF Artemia sp. as sterile food. The detailed steps in model generation and preparation of sterile live foods provide crucial technical support for constructing and applying long-term GF fish models, as well as GF Artemia sp. in microbiota-host interaction research. The protocol addresses bacterial isolation, identification, and infection on GF fish models, outlining methods for bacterial fluorescence labeling and observing their colonization in fish intestines under a microscope. GF fish, gnotobiotic fish with bacterial infection, or transferred human microbiota models will undergo various detections to elucidate their functions and effects on host immunity, digestion, behavior, transcriptomic regulation, and metabolic aspects. In the long term, this protocol can be extended to different wild-type fish species, such as marine medaka, and potentially to other selected transgenic zebrafish lines correlated to specific tissues or diseases.

Forfatter Spotlight: Etablering af kimfri zebrafisk til mikrobiota-værtsforholdsundersøgelser

Generering, vedligeholdelse og identifikation af kimfrie zebrafiskmodeller fra larver til unge stadier

The scope of this research is generation of novel, germ-free animal models, which will be helpful and powerful tools in investigation of various microbiome functions. In this field, the early germ-free fish models have been used recently, but the long-term models are still limited due to various culture conditions and the staff developmental defense. The current GF fish models are normally early life ones.Thus, the technologies including the sterile scale, the sample detection methods, and providing sterile food are very important challenges to overcome. The germ-free zebrafish model is crucial for studying host-microbiota interaction and their impacts on development and the immunity and disease. This model allows researchers to precisely manipulate the single gut bacteria and observe its effect on the transparent zebrafish embryos.It provides valuable insights into the roles of gut microbes inheriting the disease. In the future, our research will mainly focused on establishing a germ-free zebrafish model for entire life cycle to better understanding the function of the gut microbiome.

GF zebrafisk modellerne kan produceres effektivt ved at udnytte de rigelige æg gydt af par zebrafisk, med protokollen optimeret baseret på tidligere GF fisk modeller35. En enkelt 6-brønds plade kan dyrke ca. 30-48 embryoner / larver, hvilket giver mulighed for rigelig dataindsamling og statistisk analyse. Efter steril behandling dyrkes GF-embryonerne i en ren inkubator, indtil de klækkes til larver ved 48-72 timer, og ændres GZM dagligt med påvisning af indsamlede prøver, hvilket er afgøre...

Watch this Scientific Journal Video about Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies at JoVE.com

Denne protokol skitserer de primære trin for at opnå kimfrie (GF) fiskeembryoner og opretholde dem fra larver indtil ungdomsstadiet, herunder prøveudtagning og påvisning af deres sterile status. Brugen af GF-modeller med infektion er vigtig for at forstå mikrobernes rolle i værtssundheden.

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent ...

Omfanget af denne forskning er generering af nye, kimfrie dyremodeller, som vil være nyttige og kraftfulde værktøjer til undersøgelse af forskellige mikrobiomfunktioner. På dette område er de tidlige bakteriefrie fiskemodeller blevet brugt for nylig, men de langsigtede modeller er stadig begrænsede på grund af forskellige kulturforhold og personalets udviklingsforsvar. De nuværende GF-fiskemodeller er normalt tidlige i livet.Således er teknologierne, herunder den sterile skala, prøvedetekteringsmetoderne og levering af steril mad, meget vigtige udfordringer at overvinde. Den bakteriefri zebrafiskmodel er afgørende for at studere interaktion mellem vært og mikrobiota og deres indvirkning på udvikling og immunitet og sygdom. Denne model gør det muligt for forskerne præcist at manipulere de enkelte tarmbakterier og observere dens virkning på de gennemsigtige zebrafiskembryoner.Det giver værdifuld indsigt i tarmmikrobernes roller, der arver sygdommen. I fremtiden vil vores forskning primært fokusere på at etablere en bakteriefri zebrafiskmodel for hele livscyklussen for bedre at forstå tarmmikrobiomets funktion.

generation-maintenance-identification-germ-free-zebrafish-models-from

Research

JoVE Journal

Developmental Biology

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

Generation of Germ-Free Zebrafish Models from Embryos

Generering af kimfrie zebrafiskmodeller fra embryoner

GF Artemia nauplii Preparation for Chronic GF Zebrafish Nutrition

To begin, obtain 0.25 grams of Artemia cysts and rinse them with 2.4 grams per liter sodium hypochlorite for five minutes. Filter the rinsed cysts with a sterilized ultra dense filter and wash with sterilized ultrapure water for one to two minutes. After three washes, transfer them to 100 milliliters of 2.5%sterile saline and mix well.Pack 50 milliliters of treated Artemia cyst mix into a sterile 100-milliliter bottle. Using a film, seal the bottle thoroughly and place it in a shaking incubator with light. Next, using a Pasteur pipette, extract 30 milliliters of hatching Artemia nauplii from the middle and lower layers.After filtering, wash the Artemia three times with sterile ultrapure water for about one minute, and add four milliliters of sterile ultrapure water. Finally, using a disposable Pasteur pipette, retrieve actively swimming Artemia from the upper layer, wash, and prepare the solution for feeding. Microscopic observation confirmed efficient hatching of Artemia cysts after the treatment.