Questo lavoro è stato supportato da una Surgical Resident Research Scholars Program H &amp; H Lee (a gennaio), un AO Foundation Start-Up concessione S-12-03M (a LSM) e un National Institutes of Health sovvenzione R01-AI078910 (a LSM) .

<ol>
	<li>Dothager, R. 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=Advances+in+bioluminescence+imaging+of+live+animal+models.">Advances in bioluminescence imaging of live animal models.</a> Curr Opin Biotechnol. 20, 45-53 (2009).</li><li>Badr, C. E., Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+progress+and+applications.">Bioluminescence imaging progress and applications.</a> Trends Biotechnol. 29, 624-633 (2011).</li><li>Luker, G. D., Luker, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+current+applications+and+future+directions.">Optical imaging current applications and future directions.</a> J Nucl Med. 49, 1-4 (2008).</li><li>Ntziachristos, V., Ripoll, J., Wang, L. V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+listening+to+light+the+evolution+of+whole-body+photonic+imaging.">Looking and listening to light the evolution of whole-body photonic imaging.</a> Nat Biotechnol. 23, 313-320 (2005).</li><li>Reumann, M. K., Weiser, M. C., Mayer-Kuckuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Musculoskeletal+molecular+imaging+a+comprehensive+overview.">Musculoskeletal molecular imaging a comprehensive overview.</a> Trends Biotechnol. 28, 93-101 (2010).</li><li>Snoeks, T. J., Khmelinskii, A., Lelieveldt, B. P., Kaijzel, E. L., Lowik, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+advances+in+skeletal+imaging+applied+to+bone+metastases.">Optical advances in skeletal imaging applied to bone metastases.</a> Bone. 48, 106-114 (2011).</li><li>Sjollema, J., 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+potential+for+bio-optical+imaging+of+biomaterial-associated+infection+in+vivo.">The potential for bio-optical imaging of biomaterial-associated infection in vivo.</a> Biomaterials. 31, 1984-1995 (2010).</li><li>Del Pozo, J. L., Patel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Infection+associated+with+prosthetic+joints.">Clinical practice. Infection associated with prosthetic joints.</a> N Engl J Med. 361, 787-794 (2009).</li><li>Parvizi, J., Adeli, B., Zmistowski, B., Restrepo, C., Greenwald, A. S. <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+periprosthetic+joint+infection+the+current+knowledge+AAOS+exhibit+selection.">Management of periprosthetic joint infection the current knowledge AAOS exhibit selection.</a> J Bone Joint Surg Am. 94, e104 (2012).</li><li>Arciola, C. R., Campoccia, D., Speziale, P., Montanaro, L., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+in+Staphylococcus+implant+infections.+A+review+of+molecular+mechanisms+and+implications+for+biofilm-resistant+materials.">Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials.</a> Biomaterials. 33, 5967-5982 (2012).</li><li>Zimmerli, W., Moser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+treatment+concepts+of+orthopaedic+biofilm+infections.">Pathogenesis and treatment concepts of orthopaedic biofilm infections.</a> FEMS Immunol Med Microbiol. 65, 158-168 (2012).</li><li>Cram, 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=Total+knee+arthroplasty+volume+utilization+and+outcomes+among+Medicare+beneficiaries+1991-2010.">Total knee arthroplasty volume utilization and outcomes among Medicare beneficiaries 1991-2010.</a> JAMA. 308, 1227-1236 (2012).</li><li>Wolf, B. R., Lu, X., Li, Y., Callaghan, J. J., Cram, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+outcomes+in+hip+arthroplasty+long-term+trends.">Adverse outcomes in hip arthroplasty long-term trends.</a> J Bone Joint Surg Am. 94, (2012).</li><li>Bernthal, N. 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=A+mouse+model+of+post-arthroplasty+Staphylococcus+aureus+joint+infection+to+evaluate+in+vivo+the+efficacy+of+antimicrobial+implant+coatings.">A mouse model of post-arthroplasty Staphylococcus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings.</a> PLoS ONE. 5, (2010).</li><li>Bernthal, N. 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=Protective+role+of+IL-1beta+against+post-arthroplasty+Staphylococcus+aureus+infection.">Protective role of IL-1beta against post-arthroplasty Staphylococcus aureus infection.</a> J Orthop Res. 29, 1621-1626 (2011).</li><li>Niska, J. 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=Monitoring+bacterial+burden,+inflammation+and+bone+damage+longitudinally+using+optical+and+&#181;CT+imaging+in+an+orthopaedic+implant+infection+in+mice.">Monitoring bacterial burden, inflammation and bone damage longitudinally using optical and &#181;CT imaging in an orthopaedic implant infection in mice.</a> PLoS ONE. 7, e47397 (2012).</li><li>Niska, J. 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=Daptomycin+and+tigecycline+have+broader+effective+dose+ranges+than+vancomycin+as+prophylaxis+against+a+Staphylococcus+aureus+surgical+implant+infection+in+mice.">Daptomycin and tigecycline have broader effective dose ranges than vancomycin as prophylaxis against a Staphylococcus aureus surgical implant infection in mice.</a> Antimicrob Agents Chemother. 56, 2590-2597 (2012).</li><li>Niska, J. 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=Vancomycin-Rifampin+Combination+Therapy+has+Enhanced+Efficacy+Against+an+Experimental+Staphylococcus+aureus+Prosthetic+Joint+Infection.">Vancomycin-Rifampin Combination Therapy has Enhanced Efficacy Against an Experimental Staphylococcus aureus Prosthetic Joint Infection.</a> Antimicrob Agents Chemother. 57, 5080-5086 (2013).</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> J Orthop Res. 30, 335-340 (2012).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Kadurugamuwa, J. L., 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=Direct+continuous+method+for+monitoring+biofilm+infection+in+a+mouse+model.">Direct continuous method for monitoring biofilm infection in a mouse model.</a> Infect Immun. 71, 882-890 (2003).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J Biomed Opt. 12, 024007 (2007).</li><li>Kim, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+survival+and+c-kit+-progenitor+proliferation+in+Staphylococcus+aureus-infected+skin+wounds+promote+resolution.">Neutrophil survival and c-kit+-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution.</a> Blood. 117, 3343-3352 (2011).</li><li>Deirmengian, C. A., Lonner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+new+in+adult+reconstructive+knee+surgery.">What's new in adult reconstructive knee surgery.</a> J Bone Joint Surg Am. 94, 182-188 (2012).</li><li>Ning, X., 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=Maltodextrin-based+imaging+probes+detect+bacteria+in+vivo+with+high+sensitivity+and+specificity.">Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity.</a> Nat Mater. 10, 602-607 (2011).</li><li>Panizzi, 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=In+vivo+detection+of+Staphylococcus+aureus+endocarditis+by+targeting+pathogen-specific+prothrombin+activation.">In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.</a> Nat Med. 17, 1142-1146 (2011).</li><li>van Oosten, 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=Realtime+in+vivo+imaging+of+invasive+and+biomaterial+associated+bacterial+infections+using+fluorescently+labelled+vancomycin.">Realtime in vivo imaging of invasive and biomaterial associated bacterial infections using fluorescently labelled vancomycin.</a> Nat Commun. 4, 2584 (2013).</li><li>Kong, Y., 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=Imaging+tuberculosis+with+endogenous+beta+lactamase+reporter+enzyme+fluorescence+in+live+mice.">Imaging tuberculosis with endogenous beta lactamase reporter enzyme fluorescence in live mice.</a> Proc Natl Acad Sci USA. 107, 12239-12244 (2010).</li></ol>

JAM, BNT, EL, NZ, KPF sono pagati i dipendenti di PerkinElmer, che produce gli strumenti di imaging, a condizione che il Xen29 bioluminescente S. ceppo aureus, e pagato per le spese di pubblicazione di questo video-articolo. Gli altri autori non hanno nulla da rivelare.

Multimodalità di imaging come ad esempio tecniche di imaging che utilizzano in vivo imaging ottico in combinazione con l&#39;imaging μCT fornisce un nuovo approccio tecnologico che permette la visualizzazione 3D, la quantificazione e il monitoraggio longitudinale dei processi biologici in un contesto anatomico 1-4. I protocolli di questo studio forniscono informazioni dettagliate su come in vivo bioluminescenti e di imaging fluorescente può essere combinato con l&#39;imaging μCT in un impianto protesico ortopedico modello di infezione nei topi per monitorare la carica batterica, infiammazione neutrofila e cambiamenti anatomici nel midollo non invasivo e longitudinalmente su tempo. Nel loro insieme, le informazioni ottenute dalla combinazione di imaging ottico e strutturale rappresenta un importante progresso tecnologico, che può essere particolarmente adatto per lo studio dei processi biologici e patologici che colpiscono il sistema muscolo-scheletrico. Un interesseconstatazione zione che deve essere sottolineato è che abbiamo osservato che i segnali fluorescenti EGFP-neutrofili scendono a livelli di fondo da 14-21 giorni e sono rimasti a livelli di fondo per tutta la durata dell&#39;esperimento, nonostante la presenza di batteri bioluminescenti. E &#39;improbabile che l&#39;irradiazione di raggi X influenzato la sopravvivenza dei neutrofili, come abbiamo osservato cinetiche simili dei segnali neutrofili nei topi non irradiati 19. Nel nostro precedente lavoro che coinvolge un modello di S. ferite infette aureus, infiltrazione di neutrofili coinvolto una combinazione di robusta reclutamento dei neutrofili dalla circolazione, la sopravvivenza dei neutrofili prolungato nel sito di infezione e l&#39;homing delle cellule progenitrici + KIT per l&#39;ascesso, dove localmente danno luogo a maturare neutrofili 23. E &#39;probabile che processi simili hanno contribuito a infiltrazione di neutrofili nel impianto ortopedico S. modello di infezione aureus. Anche se non si sa il motivo per cui i segnali neutrofili diminuiti nel Orthopmodello di infezione aedic, potrebbe essere che la risposta immunitaria è cambiato nel corso del tempo come questa infezione progredito da una acuta infezione cronica, e questo è un argomento di indagine futura. Ci sono limitazioni con questo modello murino di infezione protesi articolare ortopedica e la multimodalità di imaging in vivo che dovrebbe essere notato. In primo luogo, questo modello di topo è una semplificazione delle procedure reali e materiali utilizzati in chirurgia ortopedica negli esseri umani 24. Tuttavia, questo modello fa ricapitolare l&#39;infezione cronica e conseguente infiammazione nel tessuto osseo e articolare che si vede nelle infezioni umane impianti ortopedici 8,9. In aggiunta, per ottenere le immagini μCT, relativamente basse dosi di raggi X irradiazione sono stati usati per ridurre al minimo eventuali effetti negativi sulla salute degli animali durante il corso dell&#39;infezione. Per una migliore risoluzione delle ossa, dosi più elevate di radiazioni a raggi X potrebbero essere utilizzati per l&#39;imaging μCT su eutanasia unnimals. Tuttavia, questo eliminerebbe la capacità di monitorare in maniera non invasiva e longitudinalmente i cambiamenti ossei per la durata degli esperimenti. In conclusione, l&#39;imaging multimodale che coinvolge l&#39;intera combinazione di imaging ottico animali in vivo con l&#39;imaging anatomico μCT ha permesso di informazioni più complete sulla infezione e risposta infiammatoria. Inoltre, queste tecniche hanno permesso la valutazione delle conseguenze della infezione e infiammazione nel tessuto osseo e delle articolazioni. I lavori futuri potrebbe approfittare di imaging multimodale per valutare l&#39;efficacia delle terapie antimicrobiche, le risposte immunitarie, patogenesi della malattia e dei cambiamenti reattivi nelle ossa, come abbiamo iniziato a indagare 14-18. Inoltre, l&#39;imaging multimodale potrebbe valutare sonde e rivelatori per diagnosticare la presenza di un&#39;infezione come precedentemente descritto in modelli animali di infezione coscia, endocardite, infettano polmonareioni e infezioni biomateriale 25-28. Infine, l&#39;uso di immagini multimodalità potrebbe essere esteso al di là di malattie infettive e utilizzato attraverso le discipline, tra cui ortopedia, reumatologia e oncologia, di indagare altre condizioni che l&#39;impatto del sistema muscolo-scheletrico, come il cancro scheletrico, malattia metastatica, fratture e artrite 5-7 .

Multimodalità tecniche di imaging preclinico che prevedono la combinazione di informazioni ottiche e anatomiche consentono la visualizzazione e il monitoraggio dei fenomeni biologici in 3D 1-4. Dal momento che l&#39;imaging μCT permette la visualizzazione squisita dell&#39;anatomia ossea, utilizzando immagini μCT in concomitanza di con imaging ottico rappresenta una combinazione unica che potrebbe essere particolarmente utile per i processi che coinvolgono l&#39;osso biologia 5-7 indagando. Un esempio potrebbe essere quello di utilizzare queste tecniche per studiare le infezioni impianti ortopedici, che rappresentano una complicazione disastroso seguito di interventi chirurgici ortopedici 8,9. Batteri biofilm si formano sugli oggetti estranei impiantati che promuovono la sopravvivenza dei batteri fungendo da barriera fisica che impedisce alle cellule immunitarie di rilevamento delle infezioni e blocca gli antibiotici di accedere ai batteri 10,11. L&#39;infezione cronica e persistente del tessuto articolare (artrite settica) und dell&#39;osso (osteomielite) induce il riassorbimento osseo che porta ad un allentamento della protesi ed eventuale guasto 8,9. Questo risultato osteolisi periprotesica è associata ad un aumento della morbilità e della mortalità 12,13. Nel nostro lavoro precedente, bioluminescenti in vivo e di imaging fluorescente è stato utilizzato insieme con i raggi X e micro-tomografia computerizzata di immagini (μCT) in un modello di infezione protesica articolare ortopedica nei topi 14-19. Questo modello ha coinvolto ponendo un titanio fili di Kirschner (filo K) in modo tale che l&#39;estremità tagliata dell&#39;impianto esteso nel ginocchio dai femori di topi 14-19. Un inoculo di Staphylococcus aureus (ceppo bioluminescente Xen29 o Xen36) è stato poi pipettati sulla superficie dell&#39;impianto in ginocchio prima che il sito chirurgico è stato chiuso 14-19. In vivo imaging ottico è stato utilizzato per rilevare e quantificare i segnali bioluminescenti, che corrispondeva alla number dei batteri nel tessuto infetto articolazioni e delle ossa 14-19. Inoltre, in vivo imaging di fluorescenza di topi LysEGFP, che possiedono neutrofili fluorescenti 20, è stato utilizzato per quantificare il numero di neutrofili che emigrarono per le articolazioni del ginocchio infette contenenti gli impianti K-wire 14,19. Infine, le modalità di imaging anatomiche, tra cui imaging ad alta risoluzione a raggi X e l&#39;imaging μCT, consentiti rispettivi 2D e 3D di immagini anatomiche dell&#39;osso colpito tutta la durata di infezione cronica, che avremmo arbitrariamente finire tipicamente tra 2 e 6 settimane postoperatorie 16 , 18. Utilizzando questo modello, l&#39;efficacia della terapia antimicrobica locale e sistemica, le risposte immunitarie protettive e alterazioni anatomiche patologiche in osso potrebbe essere valutata 14-18. In questo manoscritto, i protocolli dettagliati per le modalità di imaging ottico e μCT in questo ortopedica protesica modello di infezione articolare sono stati forniti da representatiSistema di studiare processi biologici nel contesto anatomico dell&#39;osso ve. Questi includono le procedure chirurgiche per modellare un ortopedico infezione protesi articolare nei topi, 2D e 3D in procedure di imaging ottico in vivo (per rilevare i segnali bioluminescenti batteriche e segnali neutrofili fluorescenti), μCT acquisizione di imaging e di analisi e co-registrazione di immagini ottiche 3D con il immagini μCT.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Xen36 bioluminescent Staphylococcus aureus strain</td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Bioluminescent Staphylococcus aureus strain derived from ATCC 49525 (Wright), a clinical isolate from a bacteremia patient</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Soy Agar</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>LysEGFP knockin mouse strain</td>
			<td></td>
			<td></td>
			<td>Not commercially available. This strain contains a knockin of enhanced green fluorescence protein (EGFP) into the lysozyme M gene</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products, Stamford, CT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kirschner-wire (titanium, 0.8 mm diameter)</td>
			<td>Synthes, West Chester, PA</td>
			<td>492.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire Cutter - Duracut T.C.</td>
			<td>H&#38;H Company, Ontario, Canada</td>
			<td>83-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, Deerfield, IL</td>
			<td>118718</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl 5-0 sutures (P-3 Reverse cutting)</td>
			<td>Ethicon, Summerville, NJ. Purchased through VWR International.</td>
			<td>95056-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Sustained-release Buprenorphine (5 ml - 1 mg/ml)</td>
			<td>Zoopharm, Windsor, CO</td>
			<td></td>
			<td>analgesic</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/IVISSPE?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS Spectrum Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>optical in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Quantum%20FX%20%20microCT?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Quantum FX in vivo &#956;CT system</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>&#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Living%20Image%20Software?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS SpectrumCT Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>combined optical and &#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/119240?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Living Image Software</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Image analysis software for in vivo optical imaging</td>
		</tr>
	</tbody>
</table>

combined in vivo optical and &#181;ct imaging to monitor infection, inflammation, and bone anatomy in an orthopaedic implant infection in mice

L&#39;imaging multimodale è emerso come un approccio tecnologico comune utilizzato sia nella ricerca preclinica e clinica. Tecniche avanzate che combinano in vivo imaging ottico e μCT consentono la visualizzazione di fenomeni biologici in un contesto anatomico. Queste modalità di imaging possono essere particolarmente utile per studiare le condizioni che l&#39;osso impatto. In particolare, le infezioni impianti ortopedici sono un importante problema clinico in chirurgia ortopedica. Queste infezioni sono difficili da trattare perché biofilm batterici si formano sui materiali impiantati chirurgicamente stranieri, con conseguente infiammazione persistente, osteomielite ed eventuale osteolisi dell&#39;osso che circonda l&#39;impianto, che si traduce in ultima analisi in impianto allentamento e fallimento. Qui, un modello murino di un impianto protesico ortopedico infetto è stato utilizzato che ha coinvolto il posizionamento chirurgico di un impianto con fili di Kirschner in un canale endomidollare del femore in modo tale che l&#39;estremità dell&#39;impianto eXtended nell&#39;articolazione del ginocchio. In questo modello, i topi LysEGFP, un ceppo di topi che ha neutrofili EGFP-fluorescenti, sono stati impiegati in combinazione con un bioluminescente ceppo Staphylococcus aureus, che emette luce naturale. I batteri sono stati inoculati nelle articolazioni del ginocchio dei topi prima di chiudere il sito chirurgico. In vivo bioluminescenti e di imaging fluorescente è stato utilizzato per quantificare la carica batterica e la risposta infiammatoria dei neutrofili, rispettivamente. Inoltre, l&#39;imaging μCT è stata eseguita sulla stessa topi in modo che la posizione 3D del bioluminescente e segnali ottici fluorescenti potrebbe essere co-registrate con le immagini μCT anatomiche. Per quantificare i cambiamenti nel tessuto osseo nel corso del tempo, il volume esterno delle ossa dei femori distali sono stati misurati in punti temporali specifici utilizzando un processo di segmentazione contorno a base semi-automatico. Nel loro insieme, la combinazione di immagini in vivo bioluminescenti / fluorescente con immagini μCT può essere particolarmente utile fo il monitoraggio non invasivo della infezione, risposta infiammatoria e cambiamenti anatomici in osso nel tempo.

In bioluminescenti vivo e di imaging fluorescente Nel presente studio, il protocollo è descritto per questo modello precedentemente pubblicata di un&#39;infezione protesi articolare ortopedica in topi 14-19, che comporta il posizionamento chirurgico di un impianto in titanio K-filo che si estende da un canale endomidollare nel femore nel giunto spazio di 14-19. S. aureus ceppo bioluminescente Xen29 (1 x 10 3 CFU in 2 ml PBS) è stato dispensato direttamente sulla parte superiore del titanio dell&#39;impianto fine del ginocchio prima di chiudere il sito chirurgico 16. Per visualizzare e quantificare il carico batterico e neutrofili afflusso non invasiva in topi anestetizzati LysEGFP, tutta imaging ottico animali in vivo è stata eseguita in sequenza di immagini i segnali bioluminescenti dai batteri e segnali EGFP fluorescenza dei neutrofili infiltranti utilizzando l&#39;intero animale ottica IVIS Spectrum in vivosistema di imaging in tre giorni postoperatori (ad esempio, giorni 2, 14 e 28). I segnali bioluminescenti di topi Xen29-infettati sono rimasti al di sopra dei segnali di topi sham-infettati per tutta la durata dell&#39;esperimento (Figura 1A, C), 16 di fondo. Il nostro lavoro precedente ha dimostrato che i segnali in vivo bioluminescenti strettamente approssimato del numero di ex vivo CFU isolati dal / tessuto osseo articolare e aderente agli impianti 17,18. Inoltre, i segnali EGFP fluorescenti erano più alti rispetto ai topi sham-infettati in momenti iniziali, ma si avvicinarono i livelli di fondo nel corso di infezione (Figura 2B, C) ​​16. 3D co-registrazione di segnali ottici in vivo con immagini μCT Per visualizzare i segnali ottici (segnali cioè, bioluminescenti batterica e EGFP fluorescenti) nel contesto anatomico delle articolazioni post-chirurgica del ginocchio in 3D, le immagini ottiche generate utilizzando il sistema di imaging IVIS Spectrum sono stati co-registrate con le immagini μCT generati utilizzando il sistema di imaging Quantum FX μCT. Questa co-registrazione potrebbe essere realizzato perché la camera di imaging del mouse può essere inserito o macchina per verificare che i topi erano nella stessa esatta orientazione. Per verificare questa precisione, i risultati sono stati confrontati con l&#39;acquisizione di un&#39;immagine eseguita usando l&#39;Spectrum CT-in vivo IVIS sistema di imaging che integra sia le modalità in un unico strumento senza richiedere trasferimento fisico dell&#39;animale. Per mappare i dati ottici sulle immagini μCT in 3D, abbiamo utilizzato una tomografia ottica algoritmo di ricostruzione diffusa 16. La ricostruzione 3D risultante è mostrato (filmato 1). Inoltre, l&#39;imaging μCT permesso la visualizzazione e la quantificazione dei cambiamenti conseguenti nella qualità e le dimensioni delle ossa che si sono verificati durantel&#39;infezione (Figura 2) 16. Come precedentemente riferito, il volume esterno osso del femore distale sostanzialmente aumentata nel tempo (Figura 2A) 16. Per quantificare queste modifiche, analisi di immagini 3D volumetrica è stata eseguita sulla distale 25% della superficie ossea del femore e le variazioni del volume osseo nel tempo sono stati normalizzati per il volume osseo iniziale. Il volume osseo esterno sostanzialmente aumentato nei topi infetti rispetto ai topi sham-infetti (Figura 2b) 16. L&#39;aumento del volume del femore distale esterno era probabilmente dovuta a un danno osseo causata da infezione del tessuto osseo e articolare, che sono stati osservati usando μCT di imaging e di analisi istologica 16. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig1highres.jpg" width="500" /> Figura 1 2D in vivo </strong&gt; bioluminescenti e segnali fluorescenti. S. aureus Xen29 o senza batteri (non infetti) sono stati inoculati in ginocchio dopo il posizionamento K-filo e topi LysEGFP sono stati ripresi utilizzando il sistema di imaging IVIS Spectrum 16. (A) Media in vivo segnali bioluminescenti misurata in termini di flusso totale (fotoni / sec) ± sem. (B) media in vivo EGFP segnali fluorescenti misurata in totale efficienza luminosa (fotoni / sec) / (W / cm 2) ± sem. (C) Rappresentante in bioluminescenti vivo e segnali fluorescenti sovrapposti su una fotografia in bianco e nero immagine dei topi. Il limite di rivelazione della carica batterica utilizzando imaging in vivo bioluminescente è compreso tra 1 e 2 x 10 1 x 10 3 CFU. * P &lt;0.05, † p &lt;0.01, ‡ p &lt;0,001 Xen29 topi infettati contro topi sham-infettati (t-test di Student [a due code]). Si prega dinotare che questo è una figura rappresentativa che comprende dati precedentemente pubblicati generati utilizzando Xen29 e ripreso con il sistema di imaging IVIS Lumina XR 16. <a href="https://www.jove.com/files/ftp_upload/51612/51612fig1highres.jpg" target="_blank">Cliccare qui per visualizzare una versione più grande di questa figura.</a> <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig2highres.jpg" width="500" /> Figura 2 3D μCT di imaging. S. aureus Xen29 o senza batteri (non infetti) sono stati inoculati in ginocchio dopo il posizionamento K-filo e topi sono stati ripresi utilizzando il Quantum FX in vivo del sistema μCT. (A) Rappresentante 3D rendering di μCT Xen29 topi -infected (pannelli superiori) e sham-infettati topi (pannelli inferiori). (B) Percentuale di variazione volume osseo esterno (distale il 25% dei femori) normalizzati al ti inizialeme punto (media ± SEM). * P &lt;0.05, † p &lt;0.01, ‡ p &lt;0,001 Xen29 topi infettati contro topi sham-infettati (t-test di Student [a due code]). Si prega di notare che questa è una figura rappresentativa che comprende dati precedentemente pubblicati generati utilizzando il ceppo bioluminescente S. aureus Xen29 e ripreso con la FX in vivo μCT sistema di imaging Quantum 16. <a href="https://www.jove.com/files/ftp_upload/51612/51612fig2highres.jpg" target="_blank">Cliccare qui per visualizzare una versione più grande di questa figura.</a> <a href="https://www.jove.com/files/ftp_upload/51612/Movie_1.mpg" target="_blank">Movie 1</a> . Rappresentante 3D anatomica co-registrazione dei segnali bioluminescenti Xen29 ei segnali fluorescenti EGFP-neutrofili in combinazione con le immagini μCT. Le immagini vengono ruotate sull&#39;asse verticale.

Watch this Scientific Journal Video about Combined In vivo Optical and ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice at JoVE.com

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

Combinato imaging ottico e μCT in un modello murino di infezione impianto ortopedico, utilizzando un ceppo ingegnerizzato bioluminescente di Staphylococcus aureus, purché la capacità di monitorare in maniera non invasiva e longitudinalmente la dinamica della infezione batterica, così come la risposta infiammatoria corrispondente e cambiamenti anatomici nel osso.

Combinato<em&gt; In vivo</em&gt; Optical e μCT Imaging Monitor per infezione, infiammazione, e Bone Anatomy in una infezione ortopedica impianto in topi

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

combined-vivo-optical-ct-imaging-to-monitor-infection-inflammation

Research

JoVE Journal

Medicine

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

16.9K Views.  David Geffen School of Medicine at University of California, Los Angeles (UCLA).  Combinato imaging ottico e μCT in un modello murino di infezione impianto ortopedico, utilizzando un ceppo ingegnerizzato bioluminescente di Staphylococcus aureus, purché la capacità di monitorare in maniera non invasiva e longitudinalmente la dinamica della infezione batterica, così come la risposta infiammatoria corrispondente e cambiamenti anatomici nel osso. 

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Ricerca

Medicina

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

Lavaggio broncoalveolare (BAL) di ricerca; ottenere un&#39;adeguata Yield Esempio

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Sperimentale trasporto pneumococco umano

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Tracciamento in vivo di sviluppo dell&#39;edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l&#39;utilizzo di immagini a risonanza magnetica

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Trattamento con vancomicina caricato di solfato di calcio e dell'osso autogeno in un modello di coniglio migliorata dell'infezione dell'osso

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant&#039;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

Rapida In Vivo valutazione della funzionalità di generazione i linfociti T citotossici di adiuvante per lo sviluppo di vaccini

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

La generazione delle fratture chiuse femorale in topi: un modello per studiare la guarigione ossea

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Analisi ad alta velocità effettiva dei dati di mappatura ottica tramite ElectroMapHigh-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Immunofluorescenza Etichettatura delle trappole extracellulari di neutrofilo umano e murino nel tessuto incorporato di paraffina

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...