We thank Drs. Xiaofang Wang and Katharina Hopp for their invaluable help with the animal models. This work has been supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK090728, DK058816).

Prima di avviare qualsiasi procedura con gli animali vivi, protocolli sperimentali dovranno essere approvati dalla cura degli animali e l&#39;uso comitato istituzionale (IACUC). 1. Configurazione Scanner <ol><li> Prima di iniziare, assicurarsi che il riscaldatore si trova in posizione OFF. </li><li> Selezionare il mini gradiente di imaging e 38 millimetri bobina RF e mini supporto imaging. </li><li> Nel foro centrale del supporto installare il gruppo a temperatura variabile. </li></ol> 2. Preparazione degli animali <ol><li> Per gli esperimenti di risonanza magnetica, ottenere l&#39;anestesia ottimali utilizzando isoflurano vaporizzato. Per l&#39;induzione dell&#39;anestesia, mettere animale in una camera di induzione foderato con un tessuto assorbente. Regolare il flussometro del vaporizzatore isoflurano a 2,0-2,5 L / min, e la isoflurano al 3% di ossigeno. </li><li> Rimuovere qualsiasi etichetta di metallo o altro oggetto metallico in questa fase. Applicare una pomata veterinario sugli occhi dell&#39;animale per prevenire la secchezza mentre sotto anestesia. </li><li> Una volta che l&#39;animaleha raggiunto il piano chirurgico di anestesia (cioè, perdita di recesso reflex a pizzico tep), posizionare l&#39;animale su un supporto con il suo naso inserito in un cono di naso. Impostare il flusso d&#39;aria anestesia nella sonda di 2,0-2,5 ml / min e la concentrazione isoflurano al 1,5-2,0% di ossigeno. Anestesia sarà consegnato attraverso il cono naso durante la procedura. Periodicamente regolare la concentrazione isoflurano a seconda dell&#39;età e del peso dell&#39;animale a mantenere un tasso di respirazione di ~ 40 bpm. </li><li> Utilizzare i titolari animali per garantire l&#39;animale al suo posto e impedire il movimento durante l&#39;esperimento MRI. Variare il tipo di supporto animale a seconda della regione del corpo da sottoporre a scansione. Nota: i titolari di misura dalla plastica di laboratorio (polipropilene, teflon, polistirolo, policarbonato) possono essere fatte per ospitare specifico esperimento e per adattarsi alle dimensioni degli animali (da topo neonato per 160 g di ratto). </li><li> Posizionare il termometro rettale nell&#39;animale per controllare la temperatura del corpo dell&#39;animale. Durante l&#39;experiment, tenere l&#39;animale a 35-37 ° C, in corrente di aria calda. Regolare la temperatura dell&#39;aria (30-38 ° C) e il flusso (1,200-2,000 l / h) sulla base del feedback della temperatura corporea dell&#39;animale. </li><li> Collegare un sensore di pressione respiratoria palloncino per addome dell&#39;animale per monitorare la frequenza respiratoria. </li><li> Fissare l&#39;animale al centro della bobina RF e posizionare accuratamente la bobina RF con l&#39;animale in MRI scanner. </li></ol> 3. MRI Experiment <ol><li> Tune e abbinare bobina RF prima di iniziare gli esperimenti per minimizzare la potenza RF utilizzato e per massimizzare il rapporto segnale-rumore. Per avviare l&#39;incontro / tuning: <ol><li> Aprire lo strumento di controllo spettrometro facendo clic sull&#39;icona strumenti. </li><li> Nello strumento di controllo spettrometro clic Acquisizione → Wobble. Una finestra Acq / Reco aprirà la visualizzazione della curva di oscillazione. </li><li> In alternativa, regolare i condensatori di sintonia e corrispondenza (utilizzando la messa a punto e di corrispondenza bacchette) a piccoli passi fino a quando la potenza RF riflessaè ridotto al minimo. L&#39;obiettivo è quello di vedere una curva con un minimo in corrispondenza dell&#39;asse verticale posizionato a zero sull&#39;asse orizzontale. </li><li> Quando la calibrazione della batteria è stato raggiunto con successo, premere il pulsante Stop nella finestra Acq / Reco. </li></ol></li><li> Acquisire le immagini esploratore nei tre piani ortogonali per creare immagini assiali, coronali e sagittali. Usare una sequenza di immagini veloce come Intra Porta veloce basso angolo di tiro (IG-FLASH) per acquisire le immagini da ricognizione 12. Utilizzare le immagini da ricognizione per impostare la geometria corretta per imaging effettivo. </li><li> A seconda degli obiettivi specifici di ricerca, selezionare la corretta sequenza di immagini e parametri e avviare la scansione con un semaforo. Questo calibrare canale RF, shim del magnete, impostato frequenza portante in risonanza per l&#39;acqua e regolare il guadagno del ricevitore, il tutto automaticamente. <ol><li> Per gli studi anatomici e T2 pesate, acquisire in fetta più 2D o in modalità 3D. Per ridurre il tempo per un dato esperimento risoluzione spaziale, mantenere il campo-di-view (FOV) più piccola possibile, ma abbastanza grande per evitare artefatti avvolgenti (2,56-3,2 cm). </li></ol></li><li> Mantenere il ciclo della sequenza selezionata leggermente più corto del ciclo respirazione animale dalla scelta corretta del tempo di ripetizione (TR) e / o il numero di fette. Ciò assicura che i dati vengono raccolti durante il periodo di quiete degli animali. <ol><li> Ad esempio, per le immagini addominali, mantenere la frequenza respiratoria dell&#39;animale a ~ 30 bpm; che è di circa 2.000 msec per respiro. Utilizzare un Turbo Rapid acquisizione con Enhancement Relax (RARE) Sequenza e acquisire 11-19 fette coronali, con TR / TE 1500/9 msec, fattore RARO 8 e (matrice 256 x 256, FOV 2,56 x 2,56 centimetri, spessore dello strato 0,75 millimetri) . Nota: Regolando il TR a 1.500 msec, e mantenendo la frequenza respiratoria dell&#39;animale ~ 30 bpm (2.000 msec per respiro), ci assicuriamo che i dati vengono raccolti durante il periodo di quiete degli animali. </li></ol></li><li> Dopo tutto acquisizione dell&#39;immagine è stata completata, posizionare l&#39;animale digitalizzatasul rilievo riscaldata e monitorare fino ambulatoriale. Dopo il recupero, rispedire l&#39;animale verso la gabbia e monitorati per almeno 1 ora prima di tornare alla struttura degli animali. </li></ol>

<ol>
	<li>Torres, V. E., Harris, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+dominant+polycystic+kidney+disease:+the+last+3+years.">Autosomal dominant polycystic kidney disease: the last 3 years.</a> Kidney Int. 76, 149-168 (2009).</li><li>Chapman, A. B., 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=Kidney+volume+and+functional+outcomes+in+autosomal+dominant+polycystic+kidney+disease.">Kidney volume and functional outcomes in autosomal dominant polycystic kidney disease.</a> Clinical journal of the American Society of Nephrology : CJASN. 7, 479-486 (2012).</li><li>Torres, V. E., Harris, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystic+kidney+disease:+genes,+proteins,+animal+models,+disease+mechanisms+and+therapeutic+opportunities.">Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities.</a> J Intern Med. 261, 17-31 (2007).</li><li>Hateboer, N., 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=Comparison+of+phenotypes+of+polycystic+kidney+disease+types+1+and+2+European+PKD1-PKD2+Study+Group.">Comparison of phenotypes of polycystic kidney disease types 1 and 2 European PKD1-PKD2 Study Group.</a> Lancet. 353, 103-107 (1999).</li><li>Rossetti, 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=Association+of+mutation+position+in+polycystic+kidney+disease+1+(PKD1)+gene+and+development+of+a+vascular+phenotype.">Association of mutation position in polycystic kidney disease 1 (PKD1) gene and development of a vascular phenotype.</a> Lancet. 361, 2196-2201 (2003).</li><li>Chapman, A. B., 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=Renal+structure+in+early+autosomal-dominant+polycystic+kidney+disease+(ADPKD):+The+Consortium+for+Radiologic+Imaging+Studies+of+Polycystic+Kidney+Disease+(CRISP)+cohort.">Renal structure in early autosomal-dominant polycystic kidney disease (ADPKD): The Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) cohort.</a> Kidney international. 64, 1035-1045 (2003).</li><li>Grantham, J. 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=Volume+progression+in+polycystic+kidney+disease.">Volume progression in polycystic kidney disease.</a> N Engl J Med. 354, 2122-2130 (2006).</li><li>Schrier, R. W., 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=Blood+Pressure+in+Early+Autosomal+Dominant+Polycystic+Kidney+Disease.">Blood Pressure in Early Autosomal Dominant Polycystic Kidney Disease.</a> The New England journal of medicine. , (2014).</li><li>Torres, V. E., 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=Angiotensin+Blockade+in+Late+Autosomal+Dominant+Polycystic+Kidney+Disease.">Angiotensin Blockade in Late Autosomal Dominant Polycystic Kidney Disease.</a> The New England journal of medicine. , (2014).</li><li>Wilson, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+polycystic+kidney+disease.">Mouse models of polycystic kidney disease.</a> Curr Top Dev Biol. 84, 311-350 (2008).</li><li>Happe, H., Peters, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+research+in+ADPKD:+lessons+from+animal+models.">Translational research in ADPKD: lessons from animal models.</a> Nature reviews. Nephrology. , (2014).</li><li>Frahm, J., Haase, A., Matthaei, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+NMR+imaging+of+dynamic+processes+using+the+FLASH+technique.">Rapid NMR imaging of dynamic processes using the FLASH technique.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 3, 321-327 (1986).</li><li>Bae, K. T., 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=Magnetic+resonance+imaging+evaluation+of+hepatic+cysts+in+early+autosomal-dominant+polycystic+kidney+disease:+the+Consortium+for+Radiologic+Imaging+Studies+of+Polycystic+Kidney+Disease+cohort.">Magnetic resonance imaging evaluation of hepatic cysts in early autosomal-dominant polycystic kidney disease: the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease cohort.</a> Clin J Am Soc Nephrol. 1, 64-69 (2006).</li><li>Hossack, K. F., Leddy, C. L., Johnson, A. M., Schrier, R. W., Gabow, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+findings+in+autosomal+dominant+polycystic+kidney+disease.">Echocardiographic findings in autosomal dominant polycystic kidney disease.</a> N Engl J Med. 319, 907-912 (1988).</li><li>Lumiaho, 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=Mitral+valve+prolapse+and+mitral+regurgitation+are+common+in+patients+with+polycystic+kidney+disease+type+1.">Mitral valve prolapse and mitral regurgitation are common in patients with polycystic kidney disease type 1.</a> American journal of kidney diseases : the official journal of the National Kidney Foundation. 38, 1208-1216 (2001).</li><li>Vallee, J. P., Ivancevic, M. K., Nguyen, D., Morel, D. R., Jaconi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+cardiac+MRI+in+small+animals.">Current status of cardiac MRI in small animals.</a> Magma. 17, 149-156 (2004).</li><li>Epstein, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+in+mouse+models+of+cardiac+disease.">MR in mouse models of cardiac disease.</a> NMR Biomed. 20, 238-255 (2007).</li><li>Bloomgarden, D. C., 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=Global+cardiac+function+using+fast+breath-hold+MRI:+validation+of+new+acquisition+and+analysis+techniques.">Global cardiac function using fast breath-hold MRI: validation of new acquisition and analysis techniques.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 37, 683-692 (1997).</li><li>Larson, A. C., 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=Self-gated+cardiac+cine+MRI.">Self-gated cardiac cine MRI.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 51, 93-102 (2004).</li><li>Smith, J. C., Corbin, T. J., McCabe, J. G., Bolon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+with+morphine+is+a+suitable+anaesthetic+regimen+for+embryo+transfer+in+the+production+of+transgenic+rats.">Isoflurane with morphine is a suitable anaesthetic regimen for embryo transfer in the production of transgenic rats.</a> Laboratory animals. 38, 38-43 (2004).</li><li>Ahrens, E. T., Srinivas, M., Capuano, S., Simhan, H. N., Schatten, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+embryonic+and+fetal+development+in+model+systems.">Magnetic resonance imaging of embryonic and fetal development in model systems.</a> Methods Mol Med. 124, 87-101 (2006).</li><li>Zhou, R., Pickup, S., Glickson, J. D., Scott, C. H., Ferrari, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+global+and+regional+myocardial+function+in+the+mouse+using+cine+and+tagged+MRI.">Assessment of global and regional myocardial function in the mouse using cine and tagged MRI.</a> Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 49, 760-764 (2003).</li><li>Stimpfel, T. M., Gershey, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+anesthetic+agents+for+human+safety+and+animal+recovery+surgery.">Selecting anesthetic agents for human safety and animal recovery surgery.</a> FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 5, 2099-2104 (1991).</li></ol>

The authors have nothing to disclose.

Questo manoscritto mostra la possibilità di utilizzare la risonanza magnetica UHF come strumento per la caratterizzazione fenotipica in vivo o il monitoraggio di farmaci in modelli di roditori per PKD. Descriviamo esperimenti fatti a 16,4 T con una vasta foro Avance III alta risoluzione spettrometro NMR dotata di accessori di imaging micro e mini. Lo spettrometro è stato guidato dal software di acquisizione ed elaborazione TopSpin2.0PV controllato da Paravision 5.1 software di ima...

La malattia del rene policistico (PKD) comprende un gruppo di malattie monogeniche caratterizzate dallo sviluppo di cisti renali. Tra loro ci sono autosomica dominante rene policistico (ADPKD) e autosomica recessiva rene policistico (ARPKD), che rappresentano i più comuni tipi 1,2. ADPKD, la forma più frequente di malattie ereditarie cistiche renali, si origina da mutazioni nei geni PKD1 o PKD2. Essa è caratterizzata da esordio tardivo, più cisti renali bilaterali, accompagnato da cisti extra-renale variabile, così come anomalie scheletriche cardiovascolari e muscolari. ARPKD, la maggior parte dei neonati che generalmente influenzano e bambini piccoli, è causata da mutazioni in PKHD1 ed è caratterizzata da reni ecogene dilatati e fibrosi epatica congenita 3. È importante sottolineare che, ADPKD è caratterizzata da eterogeneità, sia al gene (genica) e la mutazione (allelica) livelli, che si traduce in notevoli pla variabilità henotypic. Mutazioni nel gene PKD1 sono associati con grave quadro clinico (numerose cisti, la diagnosi precoce, l&#39;ipertensione, e ematuria), così come la progressione rapida stadio terminale della malattia renale (20 anni prima rispetto ai pazienti con mutazioni PKD2) 4. Malattia grave del fegato policistico (PLD) e anomalie vascolari possono essere associati a mutazioni in entrambi PKD1 e PKD2 5. La maggior parte delle complicanze renali di ADPKD ottengono prevalentemente in conseguenza dell&#39;espansione cisti con infiammazione e fibrosi associata. Sviluppo Cisti inizia in utero e continua attraverso il ciclo di vita del paziente. I reni normalmente mantengono la loro forma reniforme, anche se potevano raggiungere più di 20 volte il volume renale normale. La maggior parte dei pazienti presente distribuzione bilaterale di cisti renali, ma in alcuni casi particolari, cisti possono sviluppare in un modello unilaterale o asimmetrica. Un importante Challenge per nefrologi seguito i pazienti con ADPKD o terapie attuazione è la storia naturale della malattia. Durante la maggior parte del suo corso, la funzione renale rimane normale e quando la funzione renale inizia a diminuire, la maggior parte dei reni sono stati sostituiti da cisti. Quando le terapie sono implementati in fasi successive, è meno probabile che sia successo in quanto il paziente potrebbe aver già raggiunto un punto di non ritorno nella malattia renale cronica. Al contrario, quando le terapie vengono avviati nelle fasi iniziali, è difficile identificare una risposta basata esclusivamente sulla velocità di filtrazione glomerulare. Di conseguenza, il concetto di volume di rene come marker di progressione della malattia guadagnato l&#39;attenzione. Il Consorzio per Radiologic Imaging Studi di rene policistico Malattia (CRISP) studio ha dimostrato che nei pazienti affetti da ADPKD l&#39;incremento dei volumi di rene e cisti correla direttamente con deterioramento della funzione renale, sottolineando il potenziale di Volume totale del rene (TKV) come comemarcatore urrogate per la progressione della malattia 6,7. Di conseguenza, TKV è attualmente utilizzato come endpoint primario o secondario in più studi clinici per ADPKD 2,8,9. Più modelli murini comprese mutazioni spontanee e tecniche di ingegneria genetica hanno fatto luce sulla patogenesi di PKD 10,11. Modelli PKD1 o PKD2 (mutazioni in entrambe le PKD1 o PKD2) sono diventati più popolari, in quanto la malattia umana perfettamente imitare. Inoltre, i modelli di roditori con mutazioni in geni diversi geni PKD1 o PKD2 sono stati utilizzati come una piattaforma sperimentale di chiarire vie di segnalazione correlati alla malattia. Inoltre, molti di questi modelli sono stati utilizzati per testare potenziali terapie. Tuttavia, un fattore limitante in molti studi sui roditori per PKD è spesso la mancanza di metodi non invasivi efficienti per analizzare sequenzialmente i cambiamenti anatomici e funzionali nel rene. R magneticaesonance (MRI) è la tecnica di imaging attuale gold standard per seguire i pazienti ADPKD, fornire un eccellente contrasto dei tessuti molli e il dettaglio anatomico, e consentendo misure TKV. Anche se la risonanza magnetica è ben definito per l&#39;imaging anatomiche negli animali e nell&#39;uomo più grandi, di imaging piccoli roditori in vivo comporta sfide tecniche supplementari, dove la capacità di acquisire immagini ad alta risoluzione può limitarne l&#39;utilità. Con l&#39;introduzione di ultra-alto campo (UHF) MRI (7-16,4 T) e lo sviluppo di forti pendenze, è ora possibile ottenere una maggiore segnale-rumore rapporti e risoluzione spaziale di immagini MRI con una qualità diagnostica simile a quella ottenute negli uomini. Di conseguenza, l&#39;uso di UHF risonanza magnetica per imaging in vivo di piccoli modelli di roditori per PKD è diventato un potente strumento per i ricercatori.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AVANCEIII-700 (16.4 T)</td>
			<td>Bruker</td>
			<td>BH067206</td>
			<td>Wide-bore two channel multinuclear spectrometer equipped with mini and micro-imaging accessories for in vivo small rodent imaging</td>
		</tr>
		<tr>
			<td>TopSpin2.0PV&#160;</td>
			<td>Bruker</td>
			<td>H9088TA2</td>
			<td>Spectrometer processing software&#160;</td>
		</tr>
		<tr>
			<td>Paravision 5.1&#160;</td>
			<td>Bruker</td>
			<td>T10314L5</td>
			<td>Imaging sofware</td>
		</tr>
		<tr>
			<td>VTU BVT 3000 digital</td>
			<td>Bruker</td>
			<td>W1101095</td>
			<td>Temperature controller</td>
		</tr>
	</tbody>
</table>

use of ultra-high field mri in small rodent models of polycystic kidney disease for in vivo phenotyping and drug monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

In questo manoscritto, ci proponiamo di mostrare l&#39;utilità di UHF MRI come strumento per la caratterizzazione fenotipica in vivo o il monitoraggio di farmaci in modelli di roditori per PKD e altre malattie renali. Tutti gli esperimenti erano parte di protocolli sperimentali approvati dal IACUC. In vivo fenotipizzazione di piccoli modelli di roditori per PKD utilizzo UHF MRI: Tutti gli studi di imaging sono stati eseguiti su animali vivi in ane...

Watch this Scientific Journal Video about Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring at JoVE.com

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

use-ultra-high-field-mri-small-rodent-models-polycystic-kidney

Research

JoVE Journal

Medicine

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

11.4K Views.  Mayo Clinic. The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Video: Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Un In vivo Modello di roditore della contrazione indotta Lesioni e monitoraggio non invasivo di recupero

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Ricerca

Medicina

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipo spettrale allo studio anomalie cromosomiche negli esseri umani e nei topi con malattia del rene policistico

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

L&#39;uso di fibroblasti umani primari per il monitoraggio fenotipi mitocondriali nel campo della malattia di Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Valutazione della funzione cardiaca e Morfologia miocardica Utilizzando Small Animal Look-Locker Inversion Recovery (SALLI) MRI nei ratti

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI per il tracciamento cellulare

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Un possibile modello Zebrafish della malattia del rene policistico: Knockdown di<em&gt; Wnt5a</em&gt; Provoca cisti in Zebrafish Reni

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex Vivo intestinale Sacs per valutare mucosa Permeabilità in modelli di malattia gastrointestinale

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

3D Quantitative<em&gt; In Silico</em&gt; Modeling (q3DISM) di cerebrale amiloide-beta fagocitosi in roditori modelli di malattia di Alzheimer

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

In Vivo Valutazione dello sviluppo di callo di frattura durante la guarigione dell'osso nei topi utilizzando un MRI-compatibile con osteosintesi per il femore di Mouse

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...