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 ...

Postoperative infection is a devastating complication of spinal surgery. This mouse model of spinal implant infection allows researchers to study the effect of host, implant, and therapeutic factors on infection. This technique quantifies real-time longitudinal bacterial burden, humanely and efficiently, in-vivo.Using a frozen stock of S.aureus zen 36, streak the bacteria onto Luria broth plates containing 200 micrograms per milliliter of kanamycin and incubate for 12 to 16 hours at 37 degrees Celsius. At the end of the incubation, individually culture single colonies from the S.aureus zen 36 culture in tryptic soy broth containing 200 micrograms per milliliter kanamycin for 12 to 16 hours at 37 degrees Celsius and 200 revolutions per minute. The next day, dilute the culture in fresh broth at a one to 50 ratio and culture the bacteria for an additional two hours to allow the isolation of mid-logarithmic phase bacteria.At the end of the incubation, pellet the bacterial cells by centrifugation and wash the cells three times in one milliliter of fresh PBS per wash. After the last wash, measure the absorbance at 600 nanometers. The ideal optical density is 0.750.Then, re-suspend the bacteria at a one times 10 to the third colony forming units per two microliters of PBS concentration. Use clippers to remove hair from the sacrum and upper thoracic spine of the 12 week old male C-57 black six mouse. Deliver pain medicine via subcutaneous injection immediately before skin preparation and repeat in three days.Sterilize the surgical site using three alternating Betadine and alcohol wipes. After the third wipe, place the mouse in the prone position on a sterile surgical bed and maximally flex the hips. Before making an incision, confirm a lack of response to toe pinch.Palpate for the iliac crest to approximately the lumbar four vertebrae body and use a number 15 surgical scalpel blade to make a longitudinal, two centimeter skin incision. Palpate the spinous process to confirm the midline and continue the incision down to the bone. Dissect sub-periosteally on the right side of the L-4 spinous process, extending laterally to the transverse process, and pass an open absorbable braided suture size 5.0 cephalad and caudad to the L-4 body through the fascia.Using a 25-gauge spinal needle, ream the spinous process of L-4 and insert a 0.1 millimeter diameter, one centimeter long, L-shaped surgical grade stainless steel implant along the lamina, with the long arm laying cephalad. Inoculate the implant with one times 10 to the third colony forming units per two microliters of bioluminescent S.aureus zen 36, taking care to ensure that all of the solution contacts the implant. Immediately tie the suture to ensure containment of the inoculum to the implant, and use a second absorbable suture to close the skin in a running fashion.Place the mouse on a heating pad with monitoring until full recovery. Obtain postoperative radiographs to confirm the appropriate placement of the implant. To measure the bacterial burden, on the appropriate day post-infection, use clippers to remove any regrown hair from the sacrum to the upper thoracic spine and place the mouse onto a bioluminescent imaging platform.Using large bending settings with a 13 centimeter field of view, capture the bioluminescent signal for five minutes. Then, use bioluminescent imaging software to isolate a standard ovoid region of interest to quantify the mean and maximum total flux of the bioluminescence. At the appropriate experimental endpoint, after preparing the mouse as demonstrated for the surgical procedure, use a number 15 scalpel blade to sharply incise the old incision and use sterile scissors to bluntly dissect to the L-4 spinous process.When the implant can be identified, use a needle driver to gently twist and remove the implant from its position, and use sterile forceps and scissors to harvest approximately one gram of spinous process bone and soft tissue immediately surrounding the surgical implant. Place the tissue in one milliliter of PBS in a pre-weighed small conical rhino tube containing four sharp homogenizing beads and place the implant in 250 microliters of 0.3%TWEEN 80 in triptych soy broth. Weigh the tube of tissue and use a homogenizer to homogenize the sample.Vortex the implants at medium strength for two minutes, then sonicate them for 10 minutes. Dilute the implant in tissue samples one to 10, one to 101, one to 1000, and plate them on triptych soy broth plates containing 1%agar. Place the plates in an incubator at 37 degrees Celsius overnight.The following day, count the colony forming units from the implant and surrounding tissue cultures. Infected control mice demonstrate BLI signals that peak on post-infection day 10 and remain above one times 10 to the fifth photons per second per square centimeter per teridian until sacrifice, successfully modeling a chronic spinal implant infection. Mice treated with vancomycin monotherapy exhibit a significantly lower BLI signal compared to infected controls, with a two-fold reduction from post infections day 10 through 21.After post-infection day 21, there is no significant difference in BLI between the monotherapy and infected control groups. Mice treated with vancomycin-rifampin combination therapy have an even lower BLI signal that is 20 fold lower than infected controls on post-infection day 10. This significant reduction persists until post infection day 28, after which no significant difference in BLI is observed between any of the groups.Implants and surrounding tissue harvested and processed on day 35 revealed no significant differences in colony-forming units between infected control, monotherapy, and combination therapy groups. Following this procedure, the host immune response may also be studied such as by utilizing mice with fluorescently-labeled neutrophils. The development of this efficient mouse model has allowed us to identify effective novel treatments for spinal implant infection.

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Research

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Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Langsgående Evaluering av Mouse Hind Limb Bone tap etter ryggmargsskade bruke Novel, In vivo Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Klinisk testing og Spinal Cord fjerning i en mus modell for Amyotrofisk lateral sklerose (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

En Contusion modell av alvorlig ryggmargsskade i rotter

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Kombinert<em&gt; In vivo</em&gt; Optisk og μCT Imaging å overvåke infeksjoner, betennelser, og Bone Anatomy i en Ortopedisk Implant Infeksjon i Mus

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Kontrollert Cortical Impact Modell for traumatisk hjerneskade

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Intrauterin Telemetri å måle Mouse kontraktile Trykk<em&gt; I Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

En Neonatal Mouse ryggmargskompresjon Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

Stamcelle Basert Konstruert immunitet mot HIV-infeksjon i humanisert Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Nedbryting av museceller fra menneske tumorxenotransplantater forbedrer Nedstrøms Analyse av Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...