Gli autori desiderano ringraziare Keith Michel del MD Anderson Small Animal Imaging strumento per la sua assistenza tecnica con micro-CT, Edward TH Yeh, MD, per l&#39;assistenza chirurgica, e Rebecca Bartow, dottorato di ricerca, per l&#39;assistenza editoriale. Questo lavoro è stato sostenuto in parte dalla American Heart Association.

1. Adescamento della pompa osmotica <ol><li> Utilizzare tecniche sterili (es. guanti, cappa a flusso laminare) durante la preparazione delle pompe. Rimuovere la pompa e il flusso moderatore dalla confezione. Pesare la pompa vuota e registrare il peso (mg). </li><li> Riempire la pompa lentamente, per evitare di creare bolle d&#39;aria, utilizzando una piccola siringa 1cc e punta smussata 27G tubo di riempimento. Quando la soluzione raggiunge la parte superiore della pompa, stop riempimento. </li><li> Pulire l&#39;eccesso di soluzione e pesare la pompa riempita. Per la maggior parte delle soluzioni acquose, il peso in mg è lo stesso del volume in microlitri. La differenza di peso vuoto contro riempita deve essere superiore al 90% del volume medio di riempimento specificato. Se non, evacuare la pompa con la siringa e riempire. In caso di problemi tecnici si prega di fare riferimento alla sezione di risoluzione dei problemi delle istruzioni della pompa a http://www.alzet.com/products/guide_to_use/filling.html </li><li> Utilizzare un paio di pinze per rompere la flangia bianca dal Moderat flussooppure, fare attenzione a non piegare o schiacciare il tubo. Collegare un P50 catetere a una delle estremità del moderatore, che copre circa 3-4 mm. Utilizzare una siringa per riempire il catetere e attaccato moderatore e lasciare la siringa attaccata alla parte distale del catetere. </li><li> Inserire il moderatore flusso nella pompa finché il catetere è a filo con la superficie della pompa. Quindi, rimuovere la siringa dall&#39;estremità distale del catetere. Collocare la pompa in una sterile 0,9% soluzione salina o soluzione di PBS a 37 ° C incubatore per un minimo di 6 ore (preferibilmente O / N) per adescare la pompa prima di impiantarlo nel mouse. La portata della pompa varia a seconda del numero di modello (es. Modello 1002 = 0,25 ml / ora per 14 giorni). </li></ol> 2. Creazione unilaterale Ischemia degli arti posteriori del mouse <ol><li> Anestetizzare i topi con isoflurano insieme tra 2-4% a 100% O 2 ad una portata di 0,4 L / min e rimuovere i capelli (preferibilmente il giorno prima dell&#39;intervento) sull&#39;area hindlimb e indietro con un depilatory crema. </li><li> Sterilizzare tutti gli strumenti chirurgici prima di un intervento chirurgico. Posizionare il mouse nella camera di induzione dell&#39;anestesia. Utilizzare le stesse impostazioni di anestesia descritte nella sezione 2.1. </li><li> Attendere fino a quando il mouse è non risponde agli stimoli esterni e poi metterlo in posizione prona sul tavolo operatorio con un cono di naso attrezzata che offre un flusso continuo di isoflurano (1,5-2,0%). </li><li> Iniettare la dose raccomandata di anestetico (Bupivacaine; 0,25% (&lt;1 ml / kg)) presso il sito di incisione, curare la pelle con una soluzione di iodio-povidone seguito dal 70% di etanolo. Fare una incisione 4-6 mm sul retro (a lato della colonna vertebrale, nella regione scapole). Separare la membrana sottocutanea con una pinza sottile, quindi accendere il mouse nella posizione supina trattare la pelle con una soluzione di iodio-povidone seguito dal 70% di etanolo e rendere 0,8-1 cm un&#39;incisione con le forbici micro-chirurgiche sopra il ginocchio verso il mediale della coscia, seguendo il percorso dell&#39;arteria femorale. </li><li> Tunnel illato del tronco con piccolo tubo guaina (6-francese) dal mediale della coscia per l&#39;incisione sul retro. </li><li> Posizionare il mouse in posizione prona. Rimuovere la pompa osmotica adescata / catetere dalla soluzione PBS e guidare il catetere, attraverso il tubo guaina, alla coscia. </li><li> Posizionare la pompa all&#39;interno della parte posteriore e chiudere l&#39;incisione posteriore con 6-0 prolene sutura (trattare la pelle con una soluzione di iodio-povidone seguito dal 70% di etanolo). Posizionare il retro del mouse in posizione supina; rimuovere il tubo guaina, lasciando il catetere esposta attraverso l&#39;incisione sulla coscia. Fissare la gamba con nastro adesivo chirurgico. </li><li> Separare il grasso sottocutaneo del tessuto per rivelare la vascolarizzazione. Per visualizzare meglio la vascolarizzazione, utilizzare un riavvolgitore per aprire l&#39;incisione. </li><li> Pierce la guaina femorale membranosa delicatamente utilizzando pinze a punta fine per accedere al fascio vascolare. Sezionare e separare l&#39;arteria femorale dalla vena femorale e del nervo vicino all&#39;inguine, avendo cura di non provocare exemorragia eccessiva. </li><li> Posizionare due 8-0 prolene suture adiacenti per interrompere l&#39;arteria femorale 2-3 mm prossimalmente alla biforcazione femorale (l&#39;origine della femorale profonda e epigastrica arterie). </li><li> Tagliare il catetere alla lunghezza appropriata e posizionare la punta alla regione antero-laterale della coscia, fissandolo con un prolene sutura 6-0 per la fascia superficiale per garantire la somministrazione mirata di sostanza. Chiudere l&#39;incisione gamba con una sutura continua 6-0. Amministrare una iniezione sottocutanea finale di analgesico (buprenorfina, 0,1 mg / kg), trattare la pelle con una soluzione di iodio-povidone seguito dal 70% di etanolo e restituire il mouse per la gabbia. Utilizza una lampada di riscaldamento durante il recupero. </li><li> Dopo un periodo di recupero di 30 minuti, preparare il mouse per il laser Doppler per confermare la creazione di ischemia degli arti posteriori. </li><li> Monitorare l&#39;animale due volte al giorno per 5 giorni dopo l&#39;intervento. Mobilità dell&#39;animale sarà osservato per analizzare dolore; rifiuti nella gabbia servirà come una indicator di mangiare. Inoltre, durante il periodo di monitor di determinare se l&#39;animale prova dolore o disagio e dare una iniezione sottocutanea di Buprenorfina (0.1mg/kg) due volte al giorno. </li></ol> 3. Imaging Preparazione del campione <ol><li> Anestetizzare il mouse come indicato nella Sezione 2.1. Trasferire il mouse sul tavolo operatorio in posizione supina. Iniettare 1.000 UI di eparina per via sottocutanea per prevenire la coagulazione nel sistema vascolare. Mentre il mouse è attaccato al musetto, fare un&#39;incisione abbastanza grande per esporre la cavità toracica e l&#39;addome inferiore. Spostare gli organi addominali al lato sinistro dell&#39;animale per esporre l&#39;aorta addominale sottorenale (IAA). Separare con attenzione l&#39;aorta dalla vena cava inferiore. </li><li> Legare la IAA prossimale per ridurre il sanguinamento durante l&#39;inserimento del catetere. Poi, fare una piccola incisione sulla IAA. Inserire un 27 ½ G smussato ago (collegato a un catetere P10) nella IAA e fissarlo con un 6-0 sutura. </li><li> Riempire dueSiringhe 20cc con soluzione fisiologica con eparina e il 10% formalina tamponata neutra (NBF). Riempire una siringa da 5 cc con una mescola di gomma di silicone radiopaco contrasto. Mescolare i tre ingredienti di MV-composto: MV-diluente: agente MV-polimerizzazione in rapporti di lavoro dei 1:1.25:0.5. Dal momento che la miscela (Microfil) ha solo un tempo di lavoro di 20 minuti prima che l&#39;agente polimerizza, si raccomanda di non aggiungere l&#39;agente MV-indurimento fino a poco prima dell&#39;iniezione nel sistema vascolare. </li><li> Fare un taglio in atrio destro e somministrare una dose eccessiva di isoflurano per indurre l&#39;eutanasia e per consentire il drenaggio dei liquidi in eccesso. Collegare la siringa soluzione salina per la punta dell&#39;ago e cominciare a lavare il sistema vascolare per 5 min. Assicurarsi che tutto il sangue viene lavata fuori dei vasi; spegnere l&#39;isoflurano e scollegare il nosecone una volta inizia il lavaggio. Quindi, collegare la siringa formalina-riempita per il catetere di cui in (3.2) e profumato pressione per un ulteriore 5 min. Infine, l&#39;ultimo perfusione siringa, con il contrastoagente, lentamente nel mouse per 5 min. </li><li> Opzionale: Fotografia del Microfil nel sistema vascolare con una fotocamera per garantire che solo la struttura arteriosa è perfuso. </li><li> Posizionare il mouse in un contenitore riempito con il 10% NBF e conservare a 4 ° CO / N per il contrasto polimerizzazione agente. Dopo 24 ore, togliere la pelle sulla metà inferiore del tronco (distale alla legatura dell&#39;aorta addominale inferiore) utilizzando strumenti di dissezione, aggiornare la NBF 10%, e posizionare il mouse nel contenitore per altri 4 giorni a RT. </li><li> Sezionare le arti posteriori controlaterale e ischemico, e posizionare i campioni in una soluzione di acido formico (Cal-Ex II) per 48 ore per facilitare decalcificazione delle ossa. Poi, lavare i campioni con acqua corrente per 1 ora e tornare al 10% NBF e procedere con Faxitron (sistema a raggi X utilizzato per garantire l&#39;osso è decalcificata) e micro-CT. </li></ol>

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	<li>Epstein, S. E., Fuchs, S., Zhou, Y. F., Baffour, R., Kornowski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+for+enhancing+collateral+development+by+administration+of+growth+factors:+basic+principles,+early+results+and+potential+hazards.">Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards.</a> Cardiovasc. Res. 49, 532-542 (2001).</li><li>Esaki, J., Marui, A., Tabata, Y., Komeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+systems+of+angiogenic+growth+factors+for+cardiovascular+diseases.">Controlled release systems of angiogenic growth factors for cardiovascular diseases.</a> Expert Opin. Drug Deliv. 4, 635-649 (2007).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, 1267-1279 (2002).</li><li>Golomb, B. A., Dang, T. T., Criqui, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+arterial+disease:+morbidity+and+mortality+implications.">Peripheral arterial disease: morbidity and mortality implications.</a> Circulation. 114, 688-699 (2006).</li><li>Hiatt, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+therapy+for+peripheral+arterial+disease+and+claudication.">Pharmacologic therapy for peripheral arterial disease and claudication.</a> J. Vasc. Surg. 36, 1283-1291 (2002).</li><li>Hirsch, A. 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=Peripheral+arterial+disease+detection,+awareness,+and+treatment+in+primary+care.">Peripheral arterial disease detection, awareness, and treatment in primary care.</a> JAMA. 286, 1317-1324 (2001).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nat. Med. 9, 713-725 (2003).</li><li>Hirsch, A. 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=ACC/AHA+Guidelines+for+the+Management+of+Patients+with+Peripheral+Arterial+Disease+(lower+extremity,+renal,+mesenteric,+and+abdominal+aortic):+a+collaborative+report+from+the+American+Associations+for+Vascular+Surgery/Society+for+Vascular+Surgery,+Society+for+Cardiovascular+Angiography+and+Interventions,+Society+for+Vascular+Medicine+and+Biology,+Society+of+Interventional+Radiology,+and+the+ACC/AHA+Task+Force+on+Practice+Guidelines+(writing+committee+to+develop+guidelines+for+the+management+of+patients+with+peripheral+arterial+disease)--summary+of+recommendations.">ACC/AHA Guidelines for the Management of Patients with Peripheral Arterial Disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Associations for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (writing committee to develop guidelines for the management of patients with peripheral arterial disease)--summary of recommendations.</a> J. Vasc. Interv. Radiol. 17, 1383-1397 (2006).</li><li>Zagorchev, L., Mulligan-Kehoe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+vessels+in+mouse+models+of+disease.">Molecular imaging of vessels in mouse models of disease.</a> Eur. J. Radiol. 70, 305-311 (2009).</li><li>Losordo, D. W., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+and+vasculogenesis+for+ischemic+disease.+Part+I:+angiogenic+cytokines.">Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines.</a> Circulation. 109, 2487-2491 (2004).</li><li>Waters, R. E., Terjung, R. L., Peters, K. G., Annex, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+human+peripheral+arterial+occlusive+disease:+implications+for+investigation+of+therapeutic+agents.">Preclinical models of human peripheral arterial occlusive disease: implications for investigation of therapeutic agents.</a> J. Appl. Physiol. 97, 773-780 (2004).</li></ol>

Gli autori dichiarano assenza di conflitto di interessi. Tutte le procedure di animali sono stati condotti in conformità con le Linee Guida per la cura e l&#39;uso di animali da laboratorio e sono stati approvati dalla cura degli animali e del Comitato Istituzionale Usa (IACUC) presso l&#39;Università del Texas Health Science Center a Houston.

Qui vi presentiamo un metodo per farmaco / sostanza consegna osmotica in un modello murino di ischemia degli arti posteriori. Inoltre, si descrive una tecnica di fusione nei quali abbiamo utilizzato Microfil per produrre una ricostruzione 3D per analisi della rete vascolare. Il livello o la gravità di ischemia varia a seconda di dove è presentata la legatura arteriosa / escissione. Abbiamo creato una doppia legatura dell&#39;arteria femorale comune prossimale all&#39;origine femorale profo...

Malattia arteriosa periferica (PAD) è una malattia aterosclerotica che provoca insufficiente apporto di sangue nelle gambe 4. Colpisce 8-12.000.000 americani, e trattamenti in corso offre solo limitato sollievo 5,6. Agenti terapeutici innovativi che migliorano la circolazione del sangue nelle gambe non solo frenare la progressione della malattia, ma anche migliorare la qualità della vita. L&#39;incidenza di PAD è maggiore nelle persone di età superiore ai 50 anni, la terapia farmacologica in modo locale è una modalità di trattamento più auspicabile in quanto l&#39;renale ridotta funzionalità epatica e spesso osservata nei pazienti più anziani possono diminuire il metabolismo dei farmaci e aumentare gli effetti collaterali con la somministrazione sistemica. Così, abbiamo creato un modello murino di PAD per esaminare se gli agenti amministrati localmente alleviare l&#39;ischemia degli arti posteriori, promuovendo l&#39;angiogenesi e rimodellamento microvascolare. Specificamente, abbiamo utilizzato una pompa osmotica cateterizzati per fornire continuamente l&#39;agente terapeutico per ilischemica muscolo della coscia di topo. Usando il nostro sistema di consegna, siamo riusciti a mantenere le concentrazioni ottimali del farmaco per l&#39;ambiente locale, questo approccio consente di adeguata bioattività della droga, evita gli effetti collaterali sistemici, e supera lo svantaggio di limitato accesso locale di droga associata a somministrazione sistemica. Inoltre, per valutare se gli agenti amministrati localmente promuovere la rivascolarizzazione, abbiamo utilizzato fusione avanzata e tecniche di imaging ad alta definizione che consentono la quantificazione delle variazioni del microcircolo. Collettivamente, la combinazione di metodologie utilizzato in questo articolo il video è utile negli studi preclinici per aiutare nella comprensione di rivascolarizzazione farmacologicamente indotto in pazienti con PAD 7-9.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Puritan sterile cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>22-029-499</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Betadine (povidone-iodine)</td>
			<td>Fisher Scientific</td>
			<td>19-065534</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>70% Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>NC9926371</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>6-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>8709</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>8-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>2775</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Osmotic pump</td>
			<td>ALZET</td>
			<td>1002</td>
			<td>Type: Tool, 14 day release</td>
		 </tr>
		 <tr>
			<td>Vinyl catheter</td>
			<td>ALZET</td>
			<td>7760</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Heparinized saline (0.9%)</td>
			<td>Baxter</td>
			<td>2B0944</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Neutral buffered formalin</td>
			<td>Richard-Allan Scientific</td>
			<td>5705</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Microfil (silicone rubber contrast agent)</td>
			<td>Flowtech</td>
			<td>MV-112</td>
			<td>Type Reagent, Microfil White</td>
		 </tr>
		 <tr>
			<td>Cal-Ex II (formic acid solution)</td>
			<td>Fisher Scientific</td>
			<td>CS511-1D</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td>CIII</td>
			<td>7571</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td>Bupivicaine</td>
			<td>Hospira, Inc.</td>
			<td>381</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Laser Doppler perfusion imager</td>
			<td>Perimed Inc.</td>
			<td>Periscan PIM3 </td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Micro-CT imaging system</td>
			<td>GE Healthcare</td>
			<td>Explore Locus SP </td>
			<td>Type:Equipment</td>
		 </tr>
		</tbody>
 </table>

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

Ricerca preclinica in modelli animali di malattia arteriosa periferica svolge un ruolo fondamentale nel testare l&#39;efficacia di agenti terapeutici studiati per stimolare la microcircolazione. La scelta del metodo di consegna per questi agenti è importante perché la via di somministrazione colpisce profondamente la bioattività e l&#39;efficacia di questi agenti 1,2. In questo articolo, abbiamo dimostrato come amministrare localmente una sostanza in arti posteriori ischemico utilizzando una pompa osmotica cateterizzazione. Questa pompa può erogare un volume fisso di soluzione acquosa continuamente per un periodo di tempo assegnato. Abbiamo inoltre presentiamo il nostro modello murino di ischemia degli arti posteriori unilaterale indotta mediante legatura dell&#39;arteria femorale comune prossimale all&#39;origine della femorale profunda e epigastrica arterie degli arti posteriori del sinistro. Infine, si descrive la cannulazione in vivo e legatura dell&#39;aorta addominale sottorenale e perfusione della vascolarizzazione hindlimb con Microfil, un agente radiopaco colata silicone. Microfil può perfusione e riempire l&#39;intero letto vascolare (arterioso e venoso), e perché abbiamo ligato il principale canale vascolare per l&#39;uscita, l&#39;agente può essere conservato nel sistema vascolare per il futuro dell&#39;imaging ex vivo con l&#39;utilizzo di piccoli campioni di micro-CT 3 .

L&#39;animazione mostra l&#39;inserimento della pompa osmotica e chirurgia ischemia degli arti posteriori dettagliati nel protocollo. Figura 1 mostra le immagini laser Doppler della perfusione della arti posteriori, che conferma l&#39;ischemia. Dopo la fusione vascolare con Microfil, un&#39;immagine 3D micro-CT della rete vascolare mostra che Microfil può riempire efficacemente i vasi (Figura 2A), ma può verificarsi che le discontinuità (Figura 2B) a causa di vari fa...

Watch this Scientific Journal Video about Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging at JoVE.com

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Mostriamo qui il<em&gt; In vivo</em&gt; Inserimento di una pompa osmotica per la consegna locale di droga costante e la creazione di ischemia degli arti posteriori in un modello di topo. Inoltre, il sistema vascolare degli arti posteriori è perfuso con Microfil, un agente radiopaco silicone, per preparare la tomografia micro-computerizzata (micro-CT), l&#39;imaging.

Osmotica Drug Delivery per arti posteriori ischemico e la perfusione dei vasi con Microfil per Micro-Tomografia Computerizzata Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

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12.2K Views.  The Texas Heart Institute at St. Luke's Episcopal Hospital. Mostriamo qui il In vivo Inserimento di una pompa osmotica per la consegna locale di droga costante e la creazione di ischemia degli arti posteriori in un modello di topo. Inoltre, il sistema vascolare degli arti posteriori è perfuso con Microfil, un agente radiopaco silicone, per preparare la tomografia micro-computerizzata (micro-CT), l'imaging.

Video: Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Perfusione retrograda e riempimento del sistema vascolare coronarico mouse come preparazione per la Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

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Medicina

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Valutazione della solidità del grasso umano iniezione in topi nudi con Micro-Tomografia Computerizzata

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Utilizzando la tomografia micro-computerizzata per la valutazione di sviluppo del tumore e follow-up di risposta al trattamento in un modello murino di cancro ai polmoni

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Intraluminale Drug Delivery per il mouse fistola artero-venosa endotelio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Imaging fluorescente combinata vicino infrarosso e la tomografia micro-computerizzata per Direttamente Visualizzazione cerebrale tromboembolie

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Metodo di consegna diretta segmentale intra-epatico utilizzando un modello di fegato di ratto Hilar morsetto

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Metodo ottimizzato di LC-MS/MS per l'analisi di High-throughput di campioni clinici di Ivacaftor, suoi principali metaboliti e Lumacaftor nei fluidi biologici di pazienti affetti da fibrosi cistica

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Tecniche di preparazione dei tessuti per l'imaging micromografico computerizzato con contrasto di modelli cardiaci di mammiferi di grandi dimensioni con malattia cronica

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.