Questo lavoro è stato finanziato dal NIH (U54CA143837), i NIH MD Anderson Cancer Center di supporto Grants (CA016672), la Fondazione V (SAC), e di un assegno di ricerca senza restrizioni da parte della Fondazione Kanzius Research (SAC, Erie, PA). Ringraziamo Kristine Ash del Dipartimento di Oncologia Chirurgica, MD Anderson Cancer Center, per l&#39;assistenza amministrativa.

Una panoramica completa sperimentale è illustrato nella Figura 1. Ulteriori dettagli sono descritti nei passaggi 1-3 sotto. 1. Valutare RF riscaldamento di NP Colloids: AuNPs come esempio <ol><li> In generale, per ogni campione indagato NP, prima lavare più volte il campione attraverso un filtro centrifugazione con acqua deionizzata (DI) per rimuovere ioni di sfondo e contaminanti. Tutti gli ioni e contaminanti sono stati rimossi dalla sospensione AuNP quando il liquido che viene lavato fuori ha tassi di riscaldamento RF simili (HR) come acqua deionizzata. Questo processo di purificazione consente anche di alte concentrazioni di NP da ottenere. Vale la pena notare che anche se usando AuNPs in questo esempio, i principi fondamentali possono essere applicati ad altri materiali NP. </li><li> Come esempio, purificare una bottiglia da 500 ml di AuNPs disponibili in commercio di diametro 5 nm e poi sottoporli ad un campo RF 13,56 MHz di elettrico-fiELD forza 90 kV / m. <ol><li> Prendere ~ 125 ml di soluzione madre AuNP e diviso tra i sei 50 tubi filtranti centrifuga kDa. Centrifugare a 3.000 giri. per 2 min 5 sec. Rimuovere i filtri tampone e ricarica filtrati con il resto dello stock soluzione. Ripetere fino a quando tutti i 500 ml è stata filtrata. </li><li> Sostituire il buffer filtrato con un simile volume di acqua deionizzata e ripetere circa 8 volte (o fino a quando gli HR RF tampone filtrati equivalenti a acqua deionizzata). Nota, analisi UV-Vis può anche essere utilizzato per monitorare picchi di assorbimento di contaminanti. Una volta che i contaminanti tampone sono stati, pipetta circa 0,5 ml di acqua deionizzata completamente rimosso in ogni filtro e sospendere nuovamente le AuNPs pipettando ripetuta. Questo dovrebbe rimuovere completamente i AuNPs dal filtro e consentire la completa risospensione. Unire tutte e sei le sospensioni in una provetta da 15 ml Eppendorf. </li><li> Una volta che i AuNPs sono stati purificati e concentrati, analizzare il campione utilizzando ICP-OES e / o ICP-MS, UV-vis e il potenziale Zeta per i dati relativi concentration e stabilità NP, rispettivamente. SEM e / o analisi TEM può essere utilizzato anche per ottenere dati morfologici. Preparazione del campione estesa di queste tecniche possono essere trovate in letteratura 4. </li></ol></li><li> Utilizzando il sistema Kanzius RF descritto in studi precedenti 8, o derivazioni di questo sistema, posizionare un 1,3 ml cilindrica quarzo cuvetta in modo che il campo elettrico RF in aria (senza campione presente) sarebbe ~ 90 kV / m all&#39;interno della cuvetta. Per un esempio di soluzione salina normale (0,9% NaCl) il campo elettrico si ridurrebbe a ~ 1,1 kV / m. Queste sono le condizioni approssimative utilizzati per consentire raffronti tra sistemi diversi. <ol><li> Pipettare 1,3 ml di un campione di 1.000 mg / l di purificato AuNP colloide nella cuvetta di quarzo e introdurlo in RF-campo. Questo può essere fatto utilizzando un portacampioni Teflon fuoriserie. Esporre campione al campo RF per un periodo di 120 secondi o fino a quando il campione raggiunge i 70 ° C per evitare la formazione di arco elettrico o bollitura rapida. Capture i dati di imaging termico (così come aree di controllo) utilizzando una telecamera a infrarossi e software associato. Ripetere questa procedura per tre volte. </li><li> Filtrare il campione attraverso un altro filtro centrifuga 50 kDa per estrarre i AuNPs dal buffer di acqua deionizzata. Riesporre il buffer RF-campo, di nuovo tre volte. La differenza in ore tra il colloide AuNP e l&#39;accumulo acqua DI sfondo determina la HR causa delle AuNPs stessi. Aspettatevi di ottenere HRs di ~ 0,3 ° C / sec e 0,05 ° C / sec per dare un HR dipendente AuNP di circa 0,25 ° C / sec. Risospendere le restanti AuNPs dal filtro in 1,3 ml di acqua per vitro esperimenti in vivo /. </li></ol></li></ol> 2. Nanoparticelle assistita RF-ipertermia indotta: studi in vitro <ol><li> Questi studi in vitro possono essere applicate a qualsiasi tipo di tipo cellula tumorale che formano monostrati 2D. In questo esperimento utilizzare carcinoma epatocellulare umano cellule derivate Hep3B. <ol><li> Piatto ~ 50.000 cells nella parte anteriore tre pozzetti di una piastra 12 pozzetti con 1 ml di terreno di coltura. Ripetere questo 6 volte (utilizzare tre piastre per Studi NP e tre piastre come controlli). Incubare a 37,5 ° C per 24 ore prima di introdurre gli NP. Sterilizzare NP primo utilizzo BioSafety Cabinet esposizione ai raggi UV-luce per 5 min. </li><li> In ogni pozzetto introdurre 0,1 ml di una soluzione di 1,000 mg / L AuNP e lasciare per un altro 24 ore. Aggiungere 0,1 ml di acqua in ciascun pozzetto delle tre piastre delle celle di controllo e lasciare anche per 24 ore. </li><li> Dopo 24 ore sono passati, aspirare i media di cellule e lavare con PBS per rimuovere eventuali AuNPs-bound superficie. Sostituire il supporto cella. Le cellule sono ora pronti per l&#39;esposizione alle radiofrequenze. </li></ol></li><li> Posizionare ogni pacco cella 12, ben all&#39;interno della RF-campo. Attendere che le cellule si sono raffreddati a 31 ° C. Accendere il generatore di radiofrequenza ed esporre per 3,5 min. La temperatura finale del supporto cella sarà ~ 37 ° C. Spegnere il campo RF. Rimuovere le cellule e metterli in un incubatore per 24 ore prima dell&#39;analisi. <ol> &lt;li&gt; rimuovere le cellule dal termostato e mezzi cellulare aspirare. Aggiungere 1,6 ml di mezzo di cellule in ciascun pozzetto e 0,4 ml di reagente MTT. Incubare le cellule per 4 ore. Aspirare il terreno e sostituirlo con 2 ml di dimetilsolfossido (DMSO). Posizionare le piastre di celle in un rocker panchina e lasciare per 10 minuti per consentire al DMSO per solubilizzare i reagenti MTT. Infine, pipetta 100 microlitri di ciascun pozzetto in una piastra da 96 pozzetti e otticamente leggere il pozzetto a 570 nm usando un lettore di piastre, come il lettore di piastre SPECTROstar Nano. </li></ol></li></ol> 3. Nanoparticelle assistita RF-ipertermia indotta: studi in vivo <ol><li> Questi studi in vivo possono essere applicate a qualsiasi tipo di cancro che si forma tumori solidi in un modello murino ortotopico o ectopica. Questo esperimento utilizza le cellule tumorali del fegato Hep3B in un tumore BALB-C modello di topo nudo ectopica. </li><li> Nota: tutti gli esperimenti in vivo sono eseguite in conformità con tutte le linee guida, regolamenti e agenzie di regolamentazione. Inoltre, il protocollo essendo dimostrato è stato eseguito sotto la guida e l&#39;approvazione della University of Texas MD Anderson Cancer Center Institutional Animal Care ed uso commissione (IACUC). <ol><li> Crescere un numero appropriato di cellule (~ 100 k) in un pallone di coltura tissutale con il mezzo di crescita appropriato. Incubare in un incubatore a 37 ° C con 5% di CO 2 per la durata di coltura cellulare. </li><li> Trattamento di cellule con tripsina (per staccare dal pallone) e produrre una soluzione di 2 milioni di cellule per ogni 25 ml. Aggiungere una quantità uguale di Matrigel (su ghiaccio) e mescolare accuratamente per preparare la soluzione ultima iniezione. Iniettare questa soluzione nella posizione desiderata sul dorso del mouse e attendere un tempo adeguato per i tumori di crescere alla dimensione desiderata (2-4 settimane per la maggior parte delle cellule). Prima esposizione a RF, BALB-C topi nudi dovrebbero sopportare tumori solidi ectopiche 0,5-1 cm di diametro. </li></ol></li><li><ol><li> Preparare i topi da utilizzare (in questo caso BALB-C topi nudi) da anaeli sthetizing con una soluzione di chetamina e xilazina per iniezione IP. Mentre i topi sono in calo addormentato, tenerli in una camera a temperatura controllata a 37 ° C. Saranno necessari 20 topi in totale, tutti i portatori di tumori di dimensioni simili. Dieci topi saranno utilizzati in combinazione con e senza AuNPs (che è di soli iniezioni PBS), mentre i restanti 10 topi saranno divisi tra controlli RF-esposti non RF-vista e: entrambi i gruppi senza iniezioni AUNP. </li><li> Una volta adeguatamente anestetizzato, iniettare i AuNPs direttamente nel tumore con una siringa da 1 cc con un ago G 27. La soluzione AuNP dovrebbe essere ad una concentrazione di Au 200 mg / L in 0,1 ml di PBS. Dopo l&#39;iniezione, utilizzare un tampone chirurgico per assorbire il sangue e pulire il sito di iniezione con un batuffolo imbevuto di alcool. </li><li> Poi montare il mouse da trattare sulla testa di ricezione del generatore RF. Il mouse deve essere posizionato in modo che il tumore è più vicina alla testa trasmissione. Proteggere le aree che non devono essere trattati, così come sensibilitiva aree come gli occhi, le orecchie e le dita dei piedi, con nastro di rame. Assicurarsi che il nastro di rame è adeguatamente contattando il piano di messa a terra in modo che non si verifichino accumulo di cariche. Inoltre, la zona di esposizione deve avere una distanza di almeno 1 cm maggiore della dimensione del sito di trattamento desiderato. </li><li> Termocamera posizione IR in modo che il tumore e zona di trattamento sono visibili. Attivare RF per 5 min. Registrare la curva della temperatura risultante. Se la terapia raggiunge temperature superiori a 42 ° C per trattamenti fermata immediatamente. </li></ol></li><li><ol><li> Dopo l&#39;esposizione RF recuperare i topi dall&#39;anestesia in una camera calda fino a quando non sono coscienti. </li><li> Ripetere l&#39;esperimento con la stessa topi 48 ore più tardi (punti 3.3.1 - 3.4.1). </li><li> Dopo l&#39;esperimento, i topi eutanasia secondo protocolli e procedure istituzionali. Registrare il peso del tumore. Per l&#39;analisi istologica fissare i tumori con formalina e incorporarli in paraffina. Sezioni tumorali sono in genere macchiati spiritoh ematossilina e eosina e di obiettivi rilevanti per misurare l&#39;efficacia terapeutica, ad esempio Ki-67, spaccati caspasi-3, ecc. </li></ol></li></ol>

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R., Bhattacharya, R., Mukherjee, P., Curley, S. 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J., Chen, X., Parini, C., Wen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+frequency+heating+of+gold+nanoparticle+dispersions+for+non-invasive+thermal+therapies.">Low frequency heating of gold nanoparticle dispersions for non-invasive thermal therapies.</a> Nanoscale. , (2012).</li><li>Sassaroli, E., Li, K. C. P., O'Neill, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio+frequency+absorption+in+gold+nanoparticle+suspensions:+a+phenomenological+study.">Radio frequency absorption in gold nanoparticle suspensions: a phenomenological study.</a> J. Phys. D App. Phys. 45, 075303 (2012).</li><li>Worle-Knirsch, J. M., Pulskamp, K., Krug, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oops+they+did+it+again!+Carbon+nanotubes+hoax+scientists+in+viability+assays.">Oops they did it again! Carbon nanotubes hoax scientists in viability assays.</a> Nano Lett. 6, 1261 (2006).</li></ol>

Non abbiamo nulla da rivelare.

Questi protocolli consentono sperimentatore analizzare compiutamente la misura in cui nanomateriali (in questo caso AuNPs) può aumentare ipertermia indotta da radiofrequenza per il trattamento del cancro. Il primo protocollo si occupa specificamente di analizzare la produzione di calore da campioni AUNP altamente concentrato e purificato. Sebbene altri gruppi hanno riportato la produzione di calore principalmente dai buffer che le AuNPs sono sospesi e non gli stessi AuNPs 9-11, i loro sistemi RF utilizzate c...

L&#39;assorbimento di energia RF da tessuto biologico (per la loro permettività elettrica intrinseca) si traduce in temperature elevate tessuto in funzione del tempo, che conduce infine alla morte cellulare da ipertermia. Si ipotizza che l&#39;ipertermia il cancro può essere ottimizzato attraverso l&#39;utilizzo di nanomateriali mirati che internalizzano all&#39;interno della cellula tumorale e agiscono come trasduttori RF-termici, lasciando intatte le cellule normali circostanti, sane. Diversi studi hanno già dimostrato che una varietà di NP può agire fonti di calore RF come efficace che aiuti il cancro necrosi 1-4. In questi saluti, NP oro (AuNPs) 3-5, nanotubi di carbonio 1, e punti quantici 6, 7 hanno esposto le caratteristiche emozionanti quando viene utilizzato sia in vitro ed in esperimenti RF vivo. Sebbene l&#39;esatta natura del meccanismo di riscaldamento di queste NP quando esposti ad un campo RF è ancora in discussione, una serie diesperimenti fondamentali che utilizzano AuNPs ha posto grande importanza sia formato NP e stati di aggregazione. E &#39;stato dimostrato che solo AuNPs con diametro &lt;10 nm si riscalda quando esposti ad un campo RF-8. Inoltre, questo meccanismo di riscaldamento viene notevolmente attenuato quando i AuNPs vengono aggregati. Questa condizione di aggregazione è stato validato anche all&#39;interno di modelli in vitro che hanno posto l&#39;importanza su ottimizzare AuNP stabilità colloidale entro compartimenti intracellulari endolysomal di efficace terapia RF 4. Tuttavia, le tecniche ei principi sperimentali utilizzati per raccogliere e valutare questi dati possono essere problematico, soprattutto nel caso di convalida profili termici RF da colloidi NP. Diversi studi hanno dimostrato che il riscaldamento Joule dello sfondo sospensione ionica che i PN sono sospese in può essere la principale fonte di produzione di calore RF e non NP stessi 9-12. Anche se il nostro recente articolo 8 ha convalidato tegli usa delle interazioni RF nel generare calore da AuNPs di diametro inferiore a 10 nm, ci proponiamo di descrivere questi protocolli più in dettaglio in questo articolo. Abbiamo anche dimostrare i protocolli e le tecniche per valutare l&#39;efficacia di AuNPs come agenti termici hyperthermic sia in vitro e in vivo per modelli di cancro al fegato. Anche se ci concentriamo principalmente su semplici colloidi di AuNPs citrato-capped, le stesse tecniche possono essere applicate ad altri ibridi AUNP come anticorpi e complessi chemioterapia-coniugato. Aderendo a tali principi il sperimentalista spera, dovrebbe essere in grado di valutare rapidamente il potenziale per qualsiasi nanomateriale ad essere un efficace agente ipertermica termico RF-indotta.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>500 ml gold nanoparticles (5 nm)</td>
			<td>Ted Pella, INC</td>
			<td>15702-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4/-15 Centrifugal Filter Units (50 kDa)</td>
			<td>Millipore</td>
			<td>UFC805024/UFC910096</td>
			<td>(4 ml and 15 ml volumes)</td>
		</tr>
		<tr>
			<td>MEM X1 Cell Culture Media</td>
			<td>Cellgro</td>
			<td>10-101-CV</td>
			<td>(add extra nutrients as necessary)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F4135-500 ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Copper Tape</td>
			<td>Ted Pella</td>
			<td>16072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Kanzius RF System (13.56 MHZ)</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IR Camera</td>
			<td>FLIR SC 6000, FLIR Systems, Inc. (Boston, MA, USA)</td>
			<td>Contact FLIR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.3 ml Quartz Cuvette</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Teflon Sample holder with Rotary Stage</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SPECTROstar Nano Microplate reader</td>
			<td>BGM Labtech</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Applied Nanofluorescence, Houston, TX)</td>
			<td>NS1 NanoSpectralyzer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ICP-OES</td>
			<td>PerkinElmer</td>
			<td>Optima 4300 DV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern</td>
			<td>Zen 3600 Zetasizer</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

protocols for assessing radiofrequency interactions with gold nanoparticles and biological systems for non-invasive hyperthermia cancer therapy

Terapie del cancro che sono meno tossiche e invasivo rispetto ai loro omologhi esistenti sono altamente desiderabili. L&#39;uso di RF elettrici campi che penetrano in profondità nel corpo, causando minima tossicità, sono attualmente in fase di studio come mezzo vitale di terapia del cancro non invasivo. E &#39;previsto che le interazioni di energia RF con nanoparticelle internalizzate (NP) possono liberare calore che può quindi causare surriscaldamento (ipertermia) della cella, infine termina in necrosi cellulare. Nel caso di sistemi non biologici, presentiamo protocolli dettagliati relativi alla quantificazione del calore liberato dai colloidi NP altamente concentrati. Per i sistemi biologici, nel caso di esperimenti in vitro, si descrivono le tecniche e le condizioni che devono essere rispettate al fine di esporre in modo efficace le cellule tumorali a radiofrequenza senza massa artefatti di riscaldamento del materiale che oscurano in modo significativo i dati. Infine, diamo una dettagliata metodologia fo in modelli in vivo del mouse con tumori epatici ectopiche.

1. Valutare il riscaldamento RF NP colloidi: AuNPs come esempio. Dopo aver seguito la sezione 1.1 - 1.2.3 aspettano di avere una soluzione stabile, e purificato altamente concentrata su 5 nm e 10 nm AuNPs diametro. Dai 500 ml as-acquistato soluzione madre, si aspettano di ottenere almeno 4 ml di soluzione ad una concentrazione di 1000 mg / L. La differenza in ore tra i AuNPs e la soluzione tampone acqua DI sfondo a questa concentrazione dovrebbe essere ~ 0,25 ° C / sec e 0,1 ° C / sec per 5 n...

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Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy | Medicine | Video

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Descriviamo i protocolli utilizzati per studiare le interazioni di 13,56 MHz a radiofrequenza (RF) elettrici-campi con colloidi nanoparticelle d&#39;oro in entrambi i sistemi non biologici e biologici (in vitro / vivo). Queste interazioni sono studiati per applicazioni nella terapia del cancro.

Protocolli per la valutazione radiofrequenza interazioni con nanoparticelle di oro e Sistemi Biologici per la non-invasiva Ipertermia Cancer Therapy

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

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10.7K Views.  University of Texas M.D. Anderson Cancer Center.  Descriviamo i protocolli utilizzati per studiare le interazioni di 13,56 MHz a radiofrequenza (RF) elettrici-campi con colloidi nanoparticelle d'oro in entrambi i sistemi non biologici e biologici (in vitro / vivo). Queste interazioni sono studiati per applicazioni nella terapia del cancro. 

Video: Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolli di colorazione di isole pancreatiche umane

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

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Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescenza Imaging Cerenkov (CLI) per il monitoraggio Cancer Therapy

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Non invasiva di misurazione ottica del metabolismo cerebrale ed emodinamica nei neonati

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

La valutazione non invasiva della funzione endoteliale e microvascolare

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Trapianto nella camera anteriore dell&#39;occhio per longitudinale, non invasiva<em&gt; In vivo</em&gt; Imaging con Single-cella di risoluzione in tempo reale

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Imaging non invasiva e analisi di ischemia cerebrale in ratti Living Utilizzando Positron Emission Tomography con<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modello murino per l&#39;imaging non invasivo per rilevare e monitorare Ovarian Cancer Ricorrenza

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Valutazione non invasiva della efficacia di nuove terapie per patologie intestinali Utilizzando Serial Imaging endoscopica Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Valutazione non invasiva della funzione Dorsiflexor muscolare nei topi

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.

Risonanza magnetica cardiaca sensibile all'ossigenazione con manovre di respirazione vasoattiva per la valutazione non invasiva della disfunzione microvascolare coronarica