Name: inducing meningococcal meningitis serogroup c in mice via intracisternal delivery
Uploaded: 2019-11-05T14:00:00
Description: Watch this Scientific Journal Video about Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery at JoVE.com

Undersøgelserne blev delvis støttet af PRIN 2012 [tilskudsnummer 2012WJSX8K]: "Host-Microbe-interaktionsmodeller i slimhinde infektioner: udvikling af nye terapeutiske strategier" og af PRIN 2017 [2017SFBFER]: "en integreret tilgang til at tackle samspillet mellem klimatilpasning, stressende forhold og antimikrobiel resistens hos udfordrende patogener ".

Denne protokol blev gennemført for at minimere dyrenes lidelser og reducere antallet af mus i overensstemmelse med Rådets direktiv fra november 24, 1986 (86/609/EØF). In vivo forsøg rapporteret i dette studie blev godkendt af Udvalget for etisk dyrepasning og-anvendelse (prot. nummer 2, 14. december 2012) og det italienske sundhedsministerium (prot. Number 0000094-A-03/01/2013). Alle procedurer skal udføres inde i Biosafety kabinet 2 (BSC2) i et BSL2 rum, og det potentielt inficerede affald bør bortskaffes i dedike...

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I denne undersøgelse beskriver vi en forsøgsprotokol til at inducere meningokokmeningitis i voksne mus af i. cist. inokulering af meningokokbakterier. Til vores viden, ingen anden model af meningokokmeningitis er blevet udviklet i laboratorie mus inficeret af i. cist. rute tidligere er denne måde blevet udforsket for at give modeller af meningokokmeningitis i både rotte31 og kanin32. Det er velkendt, at den højeste sats af meningokoksygdom er fundet mellem små børn, ...

Neisseria meningitidis er et gram negativt β-proteobakterium begrænset til den menneskelige vært, kendt for at være en af de mest almindelige årsager til meningitis og sepsis i den menneskelige befolkning i hele verden. Det koloniserer de øvre luftveje (næse og hals) af sunde og asymptomatiske bærere (2-30% af befolkningen), men bakterien undertiden undlades forskellige vært immunforsvar og spredes fra blodbanen til hjernen forårsager en ukontrolleret lokal betændelse, kendt som meningokokmeningitis. En kombination af vært og bakterielle faktorer synes at bidrage til overgangen fra kommensal til invasiv adfærd1.
N. meningitidis er specialiseret udelukkende i menneskelig kolonisering og infektion. Det har en smal vært rækkevidde og derfor har begrænset in vivo patogenesen undersøgelser på grund af manglen på egnede dyremodeller, der gengiver den menneskelige meningokok sygdom. Som følge heraf havde det ført til grundlæggende huller i forståelsen vedrørende patogenesen af septikæmi og meningitis forårsaget af meningococcus. I de seneste årtier har udviklingen af mange in vitro-systemer gjort det muligt at identificere flere meningokokvirulens faktorer2,3,4. Selv om disse værdifulde undersøgelser gav vigtig indsigt for at forstå disse faktorers rolle for en vellykket meningokokinfektion, tillod disse modeller ikke vurdering af følgerne af bakterielle interaktioner med humorale og cellulære immunsystemet og endnu mindre med hele vævet. In vivo dyremodeller af infektion er af stor relevans samt for vurderingen af beskyttelsesgrad, der er knyttet til vaccine formuleringer. Som et humant-tropisk patogen besidder meningokok passende determinanter, der er nødvendige for en vellykket infektion, såsom overfladestrukturer (dvs. type IV-Pili og uigennemsigtige proteiner) og jern optagelses systemer til humane receptorer og transportproteiner (dvs. transferrin og lactoferrin)5,6,7 til korrekt overholdelse, overleve og invadere den menneskelige vært. Endelig, de genetiske variation evner af patogenet at unddrage sig og/eller blokere den menneskelige immunrespons yderligere bidrage til den høje art tropisme8,9. Derfor kan fraværet af specifikke værts faktorer, der er involveret i interaktionen, blokere skridt i patogenens livscyklus, hvilket skaber betydelige vanskeligheder i udviklingen af små dyremodeller, der opsummerer meningokok-livscyklussen.
I løbet af de seneste årtier er flere tilgange blevet udviklet for at forbedre vores forståelse af meningokok infektiøs cyklus. Infektioner i to dyremodeller, mus og rotter, enten intraperitonealt (IP) eller intranasalt (i.n.), blev udviklet til at gengive meningokoksygdom10,11,12,13,14 ,15,16,17. Laboratoriet mus er formentlig en af de mere alsidige dyr til inducerende eksperimentel meningokokinfektion.
Hvordan end, den IP måde i infektion fører hen til den udvikling i svær sepsis selv om sig gør ikke efterligne den naturlig vej i infektion, medens den i.n. vej i infektion var nyttig hen til vurdere meningokok patogenese, selv om sig må fremkalde Lungeinfektion før sepsis10,11,12,13,14,15,16,17.
Den IP-musemodel var medvirkende til at vurdere beskyttelsen mod meningokok-udfordringen10,11,12. Musemodellen for meningokokkolonisation baseret på i.n.-infektions vejen er blevet udviklet med spædbarn-mus, da de er mere modtagelige for meningokokker, til at reproducere en invasiv infektion, der efterligner forløbet af meningokoksygdom hos mennesker 13,14,15,16,17. Desuden, at fremme meningokokreplikation i murine vært, et stigende antal tekniske strategier blev også anvendt, herunder administration af jern til dyrene til at forbedre infektionen, brugen af høj bakteriel inokulum, mus-passaged bakteriel stamme samt ansættelse af spædbarn eller immunkompromitterede dyr værter10,13,15,18,19. Udtryk for specifikke menneskelige faktorer som CD4620 eller transferrin21 har øget følsomheden af mus til denne menneskelige-Tropic bakterie; ansættelse af den menneskelige hud xenograft model af infektion har også været nyttigt at evaluere vedhæftningsevne af meningokokker til humant endotelet22,23. Kollektivt har den seneste udvikling af humaniserede Transgene mus forbedret forståelsen af meningokok patogenesen og dens vært interaktioner.
Tidligere udviklede vi en murine model af meningokokmeningitis, hvor inokulering af bakterier blev udført i Cisterna Magna af voksne mus med mus-passaged bakterier24. Kliniske parametre og overlevelsesraten for inficerede mus viste etableringen af meningitis med egenskaber svarende til dem, der ses i den menneskelige vært, samt de mikrobiologiske og histologiske analyser af hjernen. Fra disse inficerede mus, bakterier var, også, genvundet fra blod, leveren, og milt, og bakterielle belastninger fra perifere organer korreleret med den smitsomme dosis. Denne model blev især anvendt til at evaluere virulens af en isogene mutant stamme, som er defekt i L-glutamat-transporter GltT24. For nylig, ved hjælp af vores musemodel af meningokok meningitis baseret på i. cist. rute med serogruppe C stamme 93/42862,24 og en isogene mutant defekt i CSSA -genkodning for UDP-N-acetylglucosamin 2-epimerase25, har vi analyseret rollen af eksponeret sialsyre i virksomheden af sygdom i mus.
I denne protokol beskriver vi en enkel metode til at fremkalde eksperimentel meningokokmeningitis baseret på i. cist. smitte vejen i Balb/c voksne mus. Denne metode er især nyttig til karakterisering af meningokokinfektion i en murine vært, samt til vurdering af virulens mellem vilde type reference stammer og isogene mutanter. Den intra-cisternal infektions rute sikrer fuldstændig levering af meningokokker direkte til Cisterna Magna, hvilket igen letter bakterie replikation i cerebrospinalvæsken (CSF) og inducerer meningitis med funktioner, der efterligner dem til stede i mennesker2,24,25,26.

<table>
	<tbody>
		<tr>
			<td>1,8 Skirted Cryovial With external thread</td>
			<td>Starlab</td>
			<td>E3090-6222</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>30 x 115mm</td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>F.S.T.</td>
			<td>11006-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Alarm-Thermometer</td>
			<td>TESTO</td>
			<td>9000530</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoTM Proteose Peptone</td>
			<td>BD</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro Fine syringe</td>
			<td>BD</td>
			<td>320837</td>
			<td>U-100 Insulin</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 1ml 25GA 5/8in</td>
			<td>BD</td>
			<td>300014</td>
			<td>05x16mm</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 5ml</td>
			<td>BD</td>
			<td>308062</td>
			<td>07 x 30mm</td>
		</tr>
		<tr>
			<td>BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET</td>
			<td>Bio Air s.c.r.l.</td>
			<td>Aura B3</td>
			<td></td>
		</tr>
		<tr>
			<td>BioPhotometer</td>
			<td>Eppendorf</td>
			<td>Model #6131</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle D</td>
			<td>Tecniplast</td>
			<td>D</td>
			<td>Graduated up to:400ml, Total Volume 450ml, 72x72x122mm</td>
		</tr>
		<tr>
			<td>C150 CO2 Incubator</td>
			<td>Binder</td>
			<td>9040-0078</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Body Eurostandard Type II</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td>267x207x140mm, Floor area 370cm2</td>
		</tr>
		<tr>
			<td>Cell Culture Petri Dish With Lid</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Working Volume: 5mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Microcentrifuge 5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvetta semi-micro L. Form</td>
			<td>Kartell S.p.A.</td>
			<td>01938-00</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Potassium hydrogen phosphate trihydrate</td>
			<td>Carlo erba</td>
			<td>471767</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate anhydrous ACS-for analysis</td>
			<td>Carlo Erba</td>
			<td>480141</td>
			<td>g1000</td>
		</tr>
		<tr>
			<td>Diete Standard Certificate</td>
			<td>Mucedola s.r.l.</td>
			<td>4RF21</td>
			<td>Food pellet for animal</td>
		</tr>
		<tr>
			<td>Dumont Hp Tweezers 5 Stainless Steel</td>
			<td>F.S.T. by DUMONT</td>
			<td>AGT5034</td>
			<td>0,10 x 0,06 mm tip</td>
		</tr>
		<tr>
			<td>Electronic Balance</td>
			<td>Gibertini</td>
			<td>EU-C1200</td>
			<td>Max 1200g, d=0,01g, T=-1200g</td>
		</tr>
		<tr>
			<td>Eppendorf Microcentrifuge tube safe-lock</td>
			<td>Eppendorf</td>
			<td>T3545-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Sigma-Aldrich</td>
			<td>E-6376</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>F.S.T.</td>
			<td>14084-08</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Filter Top (mini- Isolator), H-Temp with lock clamps</td>
			<td>Tecniplast</td>
			<td>1264C400SUC</td>
			<td></td>
		</tr>
		<tr>
			<td>GC agar base</td>
			<td>OXOID</td>
			<td>CM0367</td>
			<td></td>
		</tr>
		<tr>
			<td>Gillies Forceps 1 x2 teeth</td>
			<td>F.S.T.</td>
			<td>11028-15</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Glicerin RPE</td>
			<td>Carlo Erba</td>
			<td>453752</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T.</td>
			<td>11052-10</td>
			<td>Serrated Tip Width: 0.8mm</td>
		</tr>
		<tr>
			<td>Inner lid</td>
			<td>Tecniplast</td>
			<td>1264C116</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron dextran solution</td>
			<td>Sigma-Aldrich</td>
			<td>D8517-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological Safety Cabinet BH-EN and BHG Class II</td>
			<td>Faster</td>
			<td>BH-EN 2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5ml&#160;</td>
			<td>BRAND</td>
			<td>PP780751</td>
			<td>screw cap PP, grad</td>
		</tr>
		<tr>
			<td>Mouse Handling Forceps</td>
			<td>F.S.T.</td>
			<td>11035-20</td>
			<td>Serrated rubber; Gripping surface:15 x 20 mm</td>
		</tr>
		<tr>
			<td>Mucotit-F2000</td>
			<td>MERZ</td>
			<td>61846</td>
			<td>2000ml</td>
		</tr>
		<tr>
			<td>Natural Latex Gloves</td>
			<td>Medica</td>
			<td>M101</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Classic C24 Incubator Shaker</td>
			<td>PBI international</td>
			<td>C-24 Classic Benchtop Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri PS Dishes</td>
			<td>VWR</td>
			<td>391-0453</td>
			<td>90X14.2MM</td>
		</tr>
		<tr>
			<td>Pipetman Classic P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td>2-20microL</td>
		</tr>
		<tr>
			<td>Pipetman Classic P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td>20-200microL</td>
		</tr>
		<tr>
			<td>Pipetman Classim P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>200-1000microL</td>
		</tr>
		<tr>
			<td>Polyvitox</td>
			<td>OXOID</td>
			<td>SR0090A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0208</td>
			<td>250g</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0240</td>
			<td>1Kg</td>
		</tr>
		<tr>
			<td>PS Disposible forceps</td>
			<td>VWR</td>
			<td>232-0191</td>
			<td></td>
		</tr>
		<tr>
			<td>Removable Divider</td>
			<td>Tecniplast</td>
			<td>1264C812</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polypropylene Tubes</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>5ml</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MOLEKULA</td>
			<td>41272436</td>
			<td></td>
		</tr>
		<tr>
			<td>SS retainer and Polyester FilterSheet</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps</td>
			<td>F.S.T.</td>
			<td>11000-12</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Stevens Tenotomy Scissors</td>
			<td>F.S.T.</td>
			<td>14066-11</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Surgical Scissor - ToughCut</td>
			<td>F.S.T.</td>
			<td>14130-17</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Touch N Tuff disposible nitrile gloves</td>
			<td>Ansell</td>
			<td>92-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Low Temperature (ULT) Freezer</td>
			<td>Haier</td>
			<td>DW-86L288</td>
			<td>Volume= 288L</td>
		</tr>
		<tr>
			<td>Wagner Scissors</td>
			<td>F.S.T.</td>
			<td>14070-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing meningococcal meningitis serogroup c in mice via intracisternal delivery

Neisseria meningitidis (meningococcus) er en smal-vært-Range mikroorganisme, globalt anerkendt som den førende årsag til bakteriel meningitis. Meningococcus er en forbigående kolonist af humant nasopharynx på ca. 10% af raske forsøgs mennesker. Under særlige omstændigheder, det erhverver en invasiv evne til at trænge ind i slimhinderne barriere og invaderer blodbanen forårsager septikæmi. I det seneste tilfælde, fulminant sepsis kunne opstå selv uden den deraf følgende udvikling af meningitis. Omvendt, bakterier kunne dårligt formere sig i blodbanen, krydse blod-hjerne barrieren, nå det centrale nervesystem, fører til fulminant meningitis. Murine modeller af bakteriel meningitis repræsenterer et nyttigt redskab til at undersøge værten-patogen interaktioner og til at analysere patogengenetiske mekanismer, der er ansvarlige for denne dødelige sygdom. Selv om flere eksperimentelle modelsystemer er blevet evalueret i løbet af de seneste årtier, ingen af disse var i stand til at reproducere de karakteristiske patologiske hændelser af meningokoksygdom. I denne forsøgsprotokol beskriver vi en detaljeret procedure for induktion af meningokokmeningitis i en musemodel, der er baseret på bakteriernes intracisternal inokulation. De særlige tegn på menneskelig meningitis blev registreret i murine vært gennem vurdering af kliniske parametre (f. eks temperatur, kropsvægt), evaluering af overlevelsesraten, mikrobiologiske analyse og histologisk undersøgelse af hjerneskade. Ved brug af intracisternal (i. cist.) inokulum, meningokokker komplet levering direkte til Cisterna Magna, hvilket fører til en meget effektiv meningokokreplikation i hjernevæv. En 1.000-fold stigning af levedygtige optælling af bakterier er observeret i omkring 18 h. Desuden, meningokokker findes også i milten, og leveren af inficerede mus, tyder på, at leveren kan repræsentere et målorgan for meningokokreplikation.

Overlevelse af mus inficeret med N. meningitidis vildtype og isogene mutant stammer. De Neisseria meningitidis -stammer, der anvendes i disse repræsentative resultater, er Serogruppe C-reference stammen 93/4286 (ET-37) og dens isogene mutant 93/4286ΩCSSA opnået ved indsættelse af inaktivering af CSSA -genet, der koder for UDP-N-acetylglucosamin 2-epimerase, der kort i kapsel syntese locus25. For at vurdere virulens graden af CSSA...

Watch this Scientific Journal Video about Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery at JoVE.com

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Her beskriver vi en metode til at inducere meningokokmeningitis gennem en intracisternal infektions rute hos voksne mus. Vi præsenterer en trinvis protokol af meningokokinfektion fra fremstilling af inokulum til intracisternal infektion; derefter registrere dyrets overlevelse og evaluere de bakterielle belastninger i murine væv.

Inducerende Meningokokmeningitis Serogruppe C hos mus via Intracisternal levering

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

Neisseria meningitidis er en af de mest almindelige årsager til sepsis og meningitidis blandt den menneskelige befolkning over hele verden, og bakterierne er begrænset til den menneskelige vært. I denne eksperimentelle protokol vil vi beskrive den skræddersyede procedure for induktion af meningokok meningitidis i en musemodel baseret på intraokisternes inokulering af bakterier. Denne murine model gengivet de karakteristiske patologiske begivenheder af meningokok meningitis som en alvorlig sygdom hos mennesker.Så det repræsenterer et nyttigt redskab til at evaluere værten-patogen interaktion og til at analysere den patogenetiske mekanisme er ansvarlig for denne dødelige sygdom. Infektionen model kunne være nyttigt ikke kun at evaluere innovative terapeutiske strategi for at forhindre bakterier replikation direkte i CSF, men også at analysere effekten af mulige passive immunterapi mod menneskelige patogener. Teknikken kræver, at træningsfasen sikrer succes ikke kun af intracisternal inoculum, men mere generelt sygdommen induktion.Demonstrere procedurerne vil være Roberta Colicchio og Chiara Pagliuca som forskere, og Elena Scaglione som ph.d.-studerende, alle af mit laboratorium. Til at begynde, vælge en enkelt koloni fra en frisk Neisseria meningitidis kultur på gonococcal agar plade suppleret med 1% polybitox supplement og inokulere i 10 milliliter GC Bouillon. Alle de procedurer, der involverer brug af meningococci skal ske under en laminar flow bio sikkerhed terning kabinet.Dyrk bakterierne ved 37 grader Celsius i en orbital ryste inkubator på 220 RPM og holde øje med den optiske tæthed af kulturen på forskellige tidspunkter med et spektrofotometer. Dyrk kulturen, indtil den når OD600 på 0,7 svarende til den tidlige eksponentielle fase. Når denne optiske tæthed er opnået, foretage frosne bestande ved at tilføje 10% glycerol og dispensere en milliliter af kulturen i kryo hætteglas.Hætteglassene opbevares ved 80 grader Celsius indtil brug. Før infektionen, tø frosne bakterier ved stuetemperatur, derefter overføres til en centrifugerør i centrifuge i 15 minutter ved 1500xG Fjern supernatant og re-suspendere på en milliliter frisk GC Bouillon indeholder fem milligram pr kg jern dextran. Før forsøget påbegyndes, skal dyrene vejes, og kropstemperaturen evalueres.Scuff dyret fra neck-down og desinficere maven med en 70% ethanol. Ca. 2 timer før infektionen, ved hjælp af en 25 gauge nål, injicere jern dextran intraperitoneally i nederste højre kvadrant af maven. Efter bedøvelse af dyret, kontrollere for dybden af anæstesi ved at bekræfte fraværet af smerte reaktion ved klemning tåen.Efter at have placeret musen på laminar flow kabinet placere den i sternal decubitus og forsigtigt strække lemmer og halshvirvelsøjlen for at holde rygsøjlen i en lige position. For at opretholde en konsekvent bakteriel suspension, forsigtigt blande det, før du lægger det i en sprøjte med otte millimeter 30 gauge nål. Desinficere hovedet med en 70% ethanol.Placer dyret i lateral recumbency, trække ørerne ud af vejen, og flex halsen moderat. Sørg for, at midterlinjen af halsen og hovedet er i perfekt parallel position til bordpladen. Rør derefter atlasvingerne, og sørg for, at de overlapper hinanden, og føl den naturlige indrykning på midterlinjen, hvor nålen er mest tilbøjelige til at komme ind i occipital hullet.Fyld en sprøjte med en 8 millimeter 30 gauge nål med den etablerede sub-dødelige bakteriel dosis af meningococci eller jern dextran suppleret GC Bouth som kontrol i et samlet volumen på 10 mikroliter. Brug nålen til at identificere injektionspunktet og derefter injicere indholdet af sprøjten til cisterna magna af mus gennem en occipital burr hul. Kassér sprøjten og nålen sikkert efter injektionen.Placer dyret i buret under en laminar flow kabinet. Vent på opvågnen og fuld genopretning af bevægelse og fortsæt for dyrets overlevelse på de inficerede dyr, som beskrevet i protokollen. Efter bedøvelse af musen, desinficere brystet med en 70% ethanol.Ved hjælp af en 25 gauge nål trække 600 til 700 mikroliter af blod ved hjerte punktering af brysthulen. Indsamle blod i et rør, der indeholder 3,8% natriumcitrat og opbevares ved 80 grader Celsius for den senere levedygtige bakterielle celletal evaluering. Læg den aflivede mus i liggende position.Ved hjælp af saks og snævler skære pelsen langs sagittal flyet af kroppen. Med stifter, fastgør huden med pelsen på siderne af kroppen. Brug skarp saks til at afskære bughinden.Derefter med saks og engangsfæcet, punktafgifter organerne, såsom milt og lever, og sættes hver i en separat steril petriskål med en milliliter GC Bouillon suppleret med 10% glycerol. Ved hjælp af et stempel af en fem millimeter sprøjte, homogenisere organer mekanisk ved stuetemperatur i cirka to til tre minutter, indtil enkelt-celle suspension er dannet og overføre det til en scurdent kryo hætteglas. Anl. Placer straks røret på tørisen.Gør GC agar plader med antibiotika og tør dem før brug ved 37 grader Celsius i to til tre timer. Der tilberedes 10 fulde serielle fortyndinger af prøverne i GC Bouillon fra hvert homogeniseret væv. Plade dem på GC agar plader og inkubere natten over ved 37 grader Celsius med 5% kuldioxid.Med 70% ethanol, desinficere hovedet af en aflivet mus. Ved hjælp af små kirurgiske saks og en fin-tippet stålforrævk, sekkærtes pelsen og huden på det fjernede hoved, derefter fortsætte mod toppen af kraniet for klart at se suturer og til at guide åbningen af kraniet. For at åbne kraniet, indsætte spidsen af små saks gennem foramen magnum.Skær mod midten af kraniet og på tværs af midterlinjen af parietal knoglen til den modsatte side af kraniet og forsigtigt skæres langs den laterale lemmer af lambdoid sutur. Brug fint tippet skrænt, startende fra den bageste parietal hjørne, løft kraniet og trække det diagonalt opad for at opdage hjernen, og sørg for, at hjernevævet ikke er knyttet til nogen knogle i hovedet, når kraniet er løftet. Brug engangstæcesk til at fjerne bindevæv mellem kraniet og hjernen og lad ikke hjernevævet tørre ud.Brug engangskræftker til straks at placere hjernen i en petriskål med en milliliter GC Bouillon suppleret med 10% glycerol. Brug stemplet af en fem milliliter sprøjte til at homogenisere hjernen mekanisk ved stuetemperatur i cirka to til tre minutter, indtil en enkelt-celle suspension er dannet. Overfør suspensionen til et 2 milliliter sterilt rør og opbevares ved 80 grader Celsius for den senere levedygtige bakterielle celletal evaluering.Overlevelse af mus inficeret med 93/4286 wild-type eller cssA defekte Neisseria meningitidis stammer blev evalueret. Den gennemsnitlige dødelige dosis for den vilde stamme svarede til meningokokudfordringen på 10 til fjerde CFU. Mens dødeligheden 10 til 5th CFU var lig med 83,4% og 100% med 10 til 5th CFU dosis.For at opnå den median dødelige dosis for cssA defekt mutant stamme, en dosis på 10 til 8th CFU var nødvendig og mængden 1000 gange højere end den vilde type stamme. Efter injektionen, der var en hurtig stigning i vilde-type bakterier i hjernevævet nå toppen på omkring 24 timer efter infektion. I hjernen hos mus, der er inficeret med mutantstammen, faldt de levedygtige tal gradvist over tid.Også, 33.3% af mutant inficerede mus viste bakteriel clearance fra infektionsstedet, i modsætning til vilde type inficerede dyr. 48 timer efter infektionen blev cssA-defekt mutant fuldstændigt renset i milt og lever, mens de dyr, der var inficeret med den vilde stamme, udviste en vedvarende infektion. Den beskrevne eksperimentelle metode kan være nyttig til at analysere hjerneskade forbundet med denne dødelige sygdom, ved sin patologiske evaluering, såsom immunohistokæmi, immunfluorescens, og cerebral blødning analyse.

inducing-meningococcal-meningitis-serogroup-c-mice-via-intracisternal

Research

JoVE Journal

Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Oral transmission af<em&gt; Listeria monocytogenes</em&gt; I Mus via indtagelse af forurenet mad

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Dyrkning Mikroglia fra Neonatal og Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høj</sup&gt; T celler i immunsvækkede mus

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

en<em&gt; In Vitro</em&gt; Caseum Binding Assay, der forudser lægemiddelpenetration i tuberkulose-læsioner

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Intrakranielle Subarachnoidal rute for infektion for at undersøge roller af Streptococcus suis biofilm i Meningitis i en musemodel infektion

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

Kvantitativ PCR af T7 Bacteriophage fra Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Probiotiske undersøgelser i Neonatal mus ved hjælp af sonde

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...