Name: in vivo mouse model of spinal implant infection
Uploaded: 2020-06-23T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

Forfatterne ønsker å anerkjenne mottakelsen av både Pediatric Orthopedic Society of North America Biomet Spine Grant og National Institutes of Health Clinical and Translational Science Institute KL2 Grant, og HH Lee Surgical Research Grant som viktige finansieringskilder for disse forsøkene.

Alle dyr ble håndtert i strengt samsvar med god dyrepraksis som definert i de føderale forskriftene som angitt i dyrevelferdsloven (AWA), 1996-veiledningen for omsorg og bruk av forsøksdyr, PHS-retningslinjer for human omsorg og bruk av forsøksdyr, samt institusjonens retningslinjer og prosedyrer som angitt i opplæringshåndboken for dyrepleie og bruk, og alt dyrearbeid ble godkjent av University of California Los Angeles Chancellor's Animal Research Committee (ARC).
1. S. aureus ...

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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=Management+of+postprocedural+discitis.">Management of postprocedural discitis.</a> Spine Journal. 2 (4), 279-287 (2002).</li><li>Pappou, I. P., Papadopoulos, E. C., Sama, A. A., Girardi, F. P., Cammisa, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+infections+in+interbody+fusion+for+degenerative+spinal+disease.">Postoperative infections in interbody fusion for degenerative spinal disease.</a> Clinical Orthopaedics and Related Research. 444, 120-128 (2006).</li><li>Sampedro, M. F., 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=A+biofilm+approach+to+detect+bacteria+on+removed+spinal+implants.">A biofilm approach to detect bacteria on removed spinal implants.</a> Spine. 35 (12), 1218-1224 (2010).</li><li>Pull ter Gunne, A. F., Mohamed, A. S., Skolasky, R. L., van Laarhoven, C. J., Cohen, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presentation,+incidence,+etiology,+and+treatment+of+surgical+site+infections+after+spinal+surgery.">The presentation, incidence, etiology, and treatment of surgical site infections after spinal surgery.</a> Spine. 35 (13), 1323-1328 (2010).</li><li>Olsen, M. A., 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=Risk+factors+for+surgical+site+infection+in+spinal+surgery.">Risk factors for surgical site infection in spinal surgery.</a> Journal of Neurosurgery. 98, 149-155 (2003).</li><li>Ofluoglu, E. A., 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=Implant-related+infection+model+in+rat+spine.">Implant-related infection model in rat spine.</a> Archives of Orthopaedic and Trauma Surgery. 127 (5), 391-396 (2007).</li><li>Guiboux, J. P., 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=The+role+of+prophylactic+antibiotics+in+spinal+instrumentation.+A+rabbit+model.">The role of prophylactic antibiotics in spinal instrumentation. A rabbit model.</a> Spine. 23 (6), 653-656 (1998).</li><li>Stavrakis, A. I., 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=Current+Animal+Models+of+Postoperative+Spine+Infection+and+Potential+Future+Advances.">Current Animal Models of Postoperative Spine Infection and Potential Future Advances.</a> Frontiers in Medicine (Lausanne). 2, 34 (2015).</li><li>Pribaz, J. R., 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=Mouse+model+of+chronic+post-arthroplasty+infection:+noninvasive+in+vivo+bioluminescence+imaging+to+monitor+bacterial+burden+for+long-term+study.">Mouse model of chronic post-arthroplasty infection: noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study.</a> Journal of Orthopaedic Research. 30 (3), 335-340 (2012).</li><li>Bernthal, N. 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P., 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=Monitoring+bioluminescent+Staphylococcus+aureus+infections+in+living+mice+using+a+novel+luxABCDE+construct.">Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct.</a> Infection and Immunity. 68 (6), 3594-3600 (2000).</li><li>Dworsky, E. M., 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=Novel+in+vivo+mouse+model+of+implant+related+spine+infection.">Novel in vivo mouse model of implant related spine infection.</a> Journal of Orthopaedic Research. 35 (1), 193-199 (2017).</li><li>Hegde, S. 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Implantatrelaterte infeksjoner i ryggraden gir dårlige resultater for pasienter 1,2,3,4,5. I motsetning til mange andre områder i kroppen, kan infisert maskinvare i ryggraden ofte ikke fjernes på grunn av risikoen for ustabilitet og nevrologisk kompromiss. Denne unike utfordringen i innstillingen av biofilmbakterier som er resistente mot systemisk antibioti...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated from:
Benjamin V. Kelley1 
Stephen D. Zoller1 
Danielle Greig1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles
to:
Benjamin V. Kelley1 
Christopher Hamad1 
Stephen D. Zoller1 
Danielle Greig1 
Zeinab Mamouei1 
Rene Chun1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Brandon Gettleman3 
Autreen Golzar2 
Adrian Lin2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles 
3University of South Carolina School of Medicine, University of South Carolina

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated.

Erratum: In vivo Mouse Model of Spinal Implant Infection

Hensikten med denne metoden er å beskrive en ny musemodell av spinalimplantatinfeksjon (SII). Denne modellen ble designet for å gi et billig og nøyaktig verktøy for fleksibel vurdering av effekten av verts-, patogen- og/eller implantatvariabler in vivo. Testing av potensielle terapeutiske midler og behandlingsstrategier for spinalimplantatinfeksjoner i denne modellen er rettet mot å veilede forskningsutvikling før bruk i større dyremodeller og kliniske studier.
Implantatrelatert infeksjon etter ryggkirurgi er en ødeleggende komplikasjon og forekommer dessverre hos ca. 3–8 % av pasientene som gjennomgår elektiv ryggkirurgi 1,2,3,4,5 og opptil 65 % av pasientene som gjennomgår flernivå- eller revisjonskirurgi 6. Behandling av spinalimplantatinfeksjoner krever ofte flere sykehusinnleggelser, flere operasjoner og langvarig antibiotikabehandling. SIIs gir dårlige pasientutfall, inkludert nevrologisk kompromiss, funksjonshemming og økt risiko for dødelighet. Behandling av SII er ekstremt dyrt, og koster oppover $ 900,000 per pasient7.
Staphylococcus aureus er det vanligste virulente patogenet av SII 8,9,10,11. Bakterier kan frø maskinvaren direkte under operasjonen, gjennom såret i den postoperative perioden, eller senere via hematogen spredning. I nærvær av metallimplantater danner S. aureus biofilm som beskytter bakteriene mot antibiotikabehandling og immunceller. Mens fjerning av infisert maskinvare kan bidra til effektivt å utrydde en infeksjon, er dette ofte ikke mulig i ryggraden uten å forårsake destabilisering og risikere nevrologisk kompromiss12.
I fravær av eksplanting infisert maskinvare, er det nødvendig med nye tilnærminger for å forebygge, oppdage og behandle SII. Historisk sett har det vært begrensede dyremodeller av SII for effektivt å vurdere sikkerheten og effekten av nye terapier. Tidligere dyremodeller av SII krever et stort antall dyr og innsamling av datapunkter som krever avlivning inkludert kolonitelling, histologi og kultur13,14,15. Disse modellene mangler langsgående in vivo-overvåking, og gir bare ett datapunkt per dyr og er derfor dyre og ineffektive.
Tidligere arbeid som studerte en musemodell av knæprotesinfeksjon, etablerte verdien og nøyaktigheten av ikke-invasiv in vivo optisk avbildning for å overvåke infeksjonsbyrden i lengderetningen16. Påvisning av bioluminescens gjør det mulig å kvantifisere bakteriell byrde over et langsgående tidsforløp i et enkelt dyr humant, nøyaktig og effektivt. Videre har tidligere studier vist en høy korrelasjon mellom in vivo bioluminescens og CFUer som følger implantater17. Kapasiteten til å spore infeksjon over tid, har ført til en mer nyansert forståelse av implantatrelatert infeksjon. I tillegg har overvåking av langsgående infeksjon på denne måten gjort det mulig å vurdere effektiviteten av antibiotikabehandling og nye antimikrobielle stoffer nøyaktig16,17,18.
Ved hjelp av disse verktøyene utviklet og validerte vi en modell for postoperativ spinalimplantatinfeksjon. I metoden som presenteres, bruker vi et inokulum av bioluminescerende S. aureus Xen36 for å etablere en in vivo musemodell av SII for å overvåke bakteriebelastning i lengderetningen16,17,18. Denne nye modellen gir et verdifullt verktøy for effektivt å teste potensielle deteksjons-, forebyggings- og behandlingsstrategier for SII før de brukes i større dyremodeller og kliniske studier.

<table>
	<tbody>
		<tr>
			<td>Analytical Balance ME104</td>
			<td>Mettler Toledo</td>
			<td>30029067</td>
			<td>120 g capacity, 0.1 mg readability, backlit LCD, internal adjustment, metal base</td>
		</tr>
		<tr>
			<td>BD Bacto Tryptic Soy Broth</td>
			<td>Becton Dickinson (BD)</td>
			<td>BD 211825</td>
			<td>BD Bacto Tryptic Soy Broth (Soybean-Casein Digest Medium)</td>
		</tr>
		<tr>
			<td>Biomate 3S UV-VIS Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-208300</td>
			<td>Spectrophotometer; Thermo Scientific; BioMate 3S; Six-position cell holder; Spectral bandwidth: 1.8nm; Long-life xenon lamp; Store up to 40 test methods; 16L x 13W x 9 in. H; 19 lb.; 100/240V US line cord</td>
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		<tr>
			<td>Bioshield 720+ swinging bucket rotor</td>
			<td>Thermo Scientific</td>
			<td>75003183</td>
			<td>Rotor, Swinging bucket; Thermo Scientific; BIOShield 720 high speed; Capacity: 4 x 180mL (0.72L); Angle: 90 deg. ; Max. speed/RCF: 6300rpm/7188 x g; Max. radius: 16.2cm</td>
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		<tr>
			<td>Branson Ultrasonics 2510R-MTH (Sonicator)</td>
			<td>Branson Ultrasonics</td>
			<td>CPX952217R</td>
			<td>*similar model, our model is discontinued* Branson Ultrasonics MH Series Heated Ultrasonic Cleaning Bath, 120V, 0.75 gal</td>
		</tr>
		<tr>
			<td>Bullet Blender Storm Homogenizer</td>
			<td>Next Advance</td>
			<td>BBY24M</td>
			<td>The Bullet Blender Storm is the most powerful member of the Bullet Blender family. Homogenize up to 24 of your toughest samples (mouse femur, skin, cartilage, tumor, etc.) in just minutes. Air cooling&#8482; minimizes sample heat up. Uses 1.5ml screw-cap RINO&#174; tubes or snap-cap Eppendorf&#174; Safe-lock&#8482; tubes.</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Electron Microscopy Sciences</td>
			<td>66118-10</td>
			<td>The Germinator 500 is designed to decontaminate metal micro-dissecting instruments only. It is to be 
			used exclusively for research purposes. The Germinator 500 should not be used as a substitute for 
			traditional methods of terminal sterilization. Effective sterilization cannot be assured due to lack of routine 
			sterilization-efficacy monitoring methods for glass bead sterilization. The Germinator 500 has been 
			designed and built to pass the Validation of Dry Sterilizer Spore Suspension Test: USP XXIII, Part 1211.</td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026282</td>
			<td>Single 150L</td>
		</tr>
		<tr>
			<td>IVIS Lumina X5 Imaging System</td>
			<td>Perkin Elmer</td>
			<td>CLS148590</td>
			<td>The IVIS Lumina X5 high-throughput 2D optical imaging system combines high-sensitivity bioluminescence and fluorescence with high-resolution x-ray into a compact system that fits on your benchtop. With an expanded 5 mouse field of view for 2D optical imaging plus our unique line of accessories to accelerate setup and labeling, it has never been easier or faster to get robust data&#8212;and answers&#8212;on anatomical and molecular aspects of disease.</td>
		</tr>
		<tr>
			<td>MAXQ 4450 Digtial Incubating Bench Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE4450</td>
			<td>Shaker, Incubated; Thermo Scientific; Digital; MaxQ 4450; Speed 15 to 500rpm +/-1rpm; 5 deg. C above ambient to 80 deg. C; 120V 50/60Hz</td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline</td>
			<td>Fisher Bioreagents</td>
			<td>BP24384</td>
			<td>PBS, Phosphate Buffered Saline, 1X Solution, pH 7.4</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21 Centrifuge, Ventilated</td>
			<td>Thermo Scientific</td>
			<td>75002436</td>
			<td>24 x 1.5/2.0mL rotor with ClickSeal biocontainment lid</td>
		</tr>
		<tr>
			<td>SORVALL LEGEND X1R 120V Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004261</td>
			<td>Centrifuge, Benchtop; Thermo Scientific; Sorvall Legend X1R (Refrigerated), 1L capacity; Max. Speed/RCF 15,200rpm/25,830 x g; CFC-free cooling -10C to +40C; 120V 60Hz</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus - Xen36</td>
			<td>Perkin Elmer</td>
			<td>119243</td>
			<td>Staphylococcus aureus - Xen36 bioluminescent pathogenic bacteria for in vivo and in vitro drug discovery. This product was derived from a parental strain from the American Type Culture Collection, used under license. Staph. aureus-Xen36 possesses a stable copy of the Photorhabdus luminescens lux operon on the native plasmid.</td>
		</tr>
		<tr>
			<td>TUTTNAUER AUTOCLAVE 2540E 120V</td>
			<td>Heidolph Tuttnauer</td>
			<td>23210401</td>
			<td>Sterilizer, Benchtop; Heidolph; Tuttnauer; Model 2540E; Self-contained design with refillable reservoir controls water purity for sterilization; 120V 50/60Hz; 1400w. With electronic controls</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Fisher Bioreagents</td>
			<td>BP338-500</td>
			<td>Tween 80, Fisher BioReagents, Non-ionic detergent for selective protein extraction</td>
		</tr>
		<tr>
			<td>Vortex mixer VX-200</td>
			<td>Labnet Internation</td>
			<td>S0200</td>
			<td>120V touch or continuous mixer, 230V: 0 - 2,850 rpm,120V: 0 - 3,400 rpm</td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride</td>
			<td>Pfizer Injectables/Hospira</td>
			<td>00409-4888-10</td>
			<td>0.9% Sodium Chloride Injection, USP</td>
		</tr>
	</tbody>
</table>

in vivo mouse model of spinal implant infection

Spine implantatinfeksjoner gir dårlige resultater ettersom diagnosen er utfordrende og kirurgisk utryddelse er i strid med mekanisk spinalstabilitet. Hensikten med denne metoden er å beskrive en ny musemodell av spinalimplantatinfeksjon (SII) som ble opprettet for å gi et billig, raskt og nøyaktig in vivo-verktøy for å teste potensielle terapier og behandlingsstrategier for spinalimplantatinfeksjoner.
I denne metoden presenterer vi en modell av posterior-tilnærming spinalkirurgi der en rustfritt stål k-wire blir transfiksert inn i L4 spinøs prosess av 12 uker gamle C57BL / 6J villtype mus og inokulert med 1 x 103 CFU av en bioluminescerende stamme av Staphylococcus aureus Xen36 bakterier. Mus blir deretter langsgående avbildet for bioluminescens in vivo på postoperative dager 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28 og 35. Bioluminescens imaging (BLI) signaler fra et standardisert synsfelt er kvantifisert for å måle in vivo bakteriell byrde.
For å kvantifisere bakterier som fester seg til implantater og peri-implantatvev, blir mus avlivet og implantatet og omkringliggende bløtvev høstes. Bakterier løsnes fra implantatet ved sonikering, dyrket over natten og deretter telles kolonidannende enheter (CFUer). Resultatene fra denne metoden inkluderer langsgående bakterietall målt ved in vivo S. aureus bioluminescens (gjennomsnittlig maksimal fluks) og CFU-tall etter eutanasi.
Mens tidligere dyremodeller av instrumentert ryggradsinfeksjon har involvert invasiv, ex vivo vevsanalyse, utnytter musemodellen av SII presentert i dette papiret ikke-invasiv, sanntids in vivo optisk avbildning av bioluminescerende bakterier for å erstatte statisk vevsstudie. Anvendelser av modellen er brede og kan omfatte bruk av alternative bioluminescerende bakteriestammer, inkorporering av andre typer genetisk utviklede mus for samtidig å studere vertsimmunrespons, og evaluere nåværende eller undersøke nye diagnostiske og terapeutiske modaliteter som antibiotika eller implantatbelegg.

Prosedyren som presenteres her ble brukt til å vurdere effekten av antibiotikaregimer i en in vivo musemodell av SII. Spesielt ble effekten av kombinasjonsbehandling med vankomycin og rifampicin sammenlignet med vankomycin monoterapi og ubehandlede infiserte kontroller.
Før operasjonen ble mus randomisert til enten kombinasjonsbehandling, monoterapi eller infisert kontroll. En statistisk styrkeanalyse ble utført for å beregne utvalgsstørrelse. Forventede gjennomsnitt av gjennomsnittlig ma...

Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

In Vivo Mouse Model of Spinal Implant Infection

Protokollen beskriver en ny in vivo musemodell av spinalimplantatinfeksjon der et rustfritt stål k-wire implantat er infisert med bioluminescerende Staphylococcus aureus Xen36. Bakteriebelastning overvåkes longitudinelt med bioluminescerende avbildning og bekreftes med kolonidannende enhetstall etter eutanasi.

In vivo musemodell av spinalimplantatinfeksjon

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

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