Gli autori desiderano ringraziare Shawna Rideout-Gros e Garces Alexandra per il loro aiuto con la cura degli animali, e Wu Ping per la sua assistenza tecnica. Il supporto per questo lavoro è stato fornito dal National Institutes of Health in concessione n. EB003268, e il Canada Research Chairs programma.

1. L&#39;ecografia e la risonanza magnetica Setup Una risonanza magnetica compatibile con tre assi ultrasuoni focalizzati è stato utilizzato in questo studio (FUS Instruments, Inc., Toronto, Ontario, Canada). Due trasduttori di ultrasuoni sono stati utilizzati differenti: uno in casa costruita 551,5 kHz sfericamente focalizzato trasduttore (raggio di curvatura = 60 mm, diametro esterno = 75 mm, diametro interno = 20 mm), e un 1,503 MHz, 8-settore matrice con integrato PZT idrofono (Imasonic Inc., Voray-sur-L&#39;Orgnon, Francia), guidato come un unico elemento trasduttore sferico concentrato (FN = 0.8, apertura = 10 cm). Una risonanza magnetica compatibile PVDF ricevitore 8 è stato utilizzato per registrare le emissioni acustiche quando il trasduttore 551,5 kHz è stato utilizzato. Se si utilizza attrezzature diverse, vengono proposte le seguenti: <ol><li> Selezionare un appropriato, trasduttore ad ultrasuoni calibrato con una frequenza centrale nella gamma di 0,25 MHz -1,5 MHz e un numero f (raggio di curvatura / apertura) di 1 o meno. </li><li> Posizionare un bagno d&#39;acqua firiempito con acqua calda, degasato, acqua deionizzata sul letto di un 1,5 T e 3 T MRI. Il bagno d&#39;acqua dovrebbe avere un top-plate che può contenere gli animali. Montare il trasduttore di ultrasuoni nel serbatoio in un MRI compatibile tre assi fasi di posizionamento o sistema 9, rivolta verso la superficie dell&#39;acqua. </li><li> Eseguire il cavo del trasduttore alle apparecchiature di guida ad ultrasuoni attraverso un pannello di penetrazione. </li><li> Generare il segnale di pilotaggio del trasduttore utilizzando un generatore di funzione e amplificatore di potenza. Utilizzare un circuito esterno corrispondenza di minimizzare la potenza riflessa elettrica. </li><li> Conoscendo la lunghezza focale del trasduttore, posizionare il trasduttore di messa a fuoco sulla superficie dell&#39;acqua e sonicare la superficie dell&#39;acqua per generare una fontana di acqua visibile. Fasi 1.6 e 1.7 descrivono come registrare la messa a fuoco utilizzando la fontana d&#39;acqua generato. Un metodo più preciso per localizzare la messa a fuoco, che comporta uno sonicazione gel di assorbimento di calore e di imaging dell&#39;aumento di temperatura risultante al fuoco con MRI, può essere trovato in 9 &lt;/ Sup&gt;. </li><li> Segnare la posizione della messa a fuoco con un pennarello che sarà visibile sulle immagini RM. Ciò può essere fatto utilizzando una piastra con un foro centrale che si riempie di acqua viene posizionato sopra il fuoco. L&#39;acqua-riempita foro sarà visibile in MRI e fornirà coordinate focali in due piani, mentre la coordinata nella direzione assiale del trasduttore può essere determinata dalla superficie dell&#39;acqua. </li><li> Utilizzo di un 3-piano sequenza localizzatore, l&#39;immagine del marcatore fuoco e registrare la posizione del fuoco nel sistema di coordinate MRI. </li><li> Per controllare le emissioni acustiche, montare un MR-compatibile 8 idrofoni che viene utilizzata come rivelatore cavitazione passiva (PCD) in bagno d&#39;acqua diretta al fuoco trasduttore, o utilizzare un trasduttore con idrofono integrato. </li><li> Far passare i cavi PCD su una scheda di ambito che è sufficientemente veloce per catturare raffiche fino a 10 ms a PRFs fino a 2 Hz (ad esempio ATS460, AlazarTech, Pointe-Claire, Quebec, Canada). I cavi devono essere messi a terra al penestrazione pannello o schermatura RF per minimizzare il rumore. </li></ol> 2. Preparazione degli animali <ol><li> Anestetizzare gli animali che utilizzano gas isofluorano. Poiché isofluorano ha un effetto sulla disgregazione BBB 10, gli animali devono essere tolti i gas 10 minuti prima dell&#39;inizio dell&#39;esperimento. Luogo unguento lubrificante oculare in ciascun occhio all&#39;inizio di anestesia per proteggere la cornea da essiccazione o altre lesioni. </li><li> L&#39;utilizzo di un rasoio elettrico accorciare il pelo dalla parte superiore della testa dell&#39;animale e del collo, quindi rimuovere il pelo rimanente utilizzando una crema depilatoria (ad esempio Nair, Church &amp; Dwight Co., Princeton, NJ, USA) e risciacquare il cuoio capelluto con sapone neutro e acqua. </li><li> Preparare la coda con uno scrub della pelle betadine seguito da un lavaggio bridine. Eseguire un Wipedown finale con l&#39;alcol, al fine di visualizzare la vena della coda. </li><li> Inserire un catetere calibro 22 con 3-rubinetto a tre vie nella vena della coda e lavare con una miscela di eparina / soluzione salina (33 U / mL) per prevenire la formazione del coagulonel catetere. </li><li> Fornire anestetici iniettabili ketamina (40-50 mg / kg, 10 mg / kg xilazina) tramite iniezione intramuscolare, e rimuovere l&#39;animale dal isofluorano. </li><li> Posizionare il supina anestetizzato animale sul sistema di posizionamento ultrasuoni con la sommità della testa contatto bagnomaria attraverso un foro nella piastra superiore (Fig.1). Una piccola quantità di gel ultrasuoni forse applicato alla parte superiore della testa dell&#39;animale per ridurre al minimo il rischio di bolle d&#39;aria intrappolate. </li><li> Nastro le gambe per il sistema di posizionamento. La testa può essere tenuta in posizione utilizzando una barra di morso, se disponibile, o nastro adesivo collocato saldamente in tutto il mento. </li><li> Coprire l&#39;animale con un asciugamano e una coperta circolazione acqua per mantenerlo caldo. </li></ol> 3. Obiettivo di selezione <ol><li> Acquisire basale assiale T 2-pesate e T 1-pesate immagini RM del cervello. Se si utilizza una risonanza magnetica 1,5 T e dedicato la testa di dimensioni ricezione RF-bobina di superficie, adatto scun parametri potrebbero essere: T 2 ponderata: FSE, TE = 60 ms, TR = 2000 ms T 1 pesata: FSE, TE = 10 ms, TR = 500 ms </li><li> Selezionare il bersaglio dalla T 2-pesate, evitando i ventricoli e la linea mediana del cervello, e selezionando un mesencefalo profondità. </li><li> Spostare lo stato attivo trasduttore nel percorso di destinazione. </li></ol> 4. Preparazione microbolle Microbolle DEFINITY (Lantheus Medical Imaging, MA, USA) sono utilizzati da diversi gruppi per FUS mediate microbolle indotte BBBD 2,5,7. Dosaggi appropriati per gli altri tipi di microbolle possono trovare nella letteratura 11,12. <ol><li> Attivare le microbolle DEFINITY e lentamente elaborare un piccolo volume in una siringa da 1 ml con un ago calibro 18. </li><li> Rimuovere aria intrappolata dalla siringa delicatamente spostando lo stantuffo avanti e indietro. Non toccare la siringa in quanto può rompere le bolle. </li><li> Diluire il Definity nella normale<html> soluzione salina in un rapporto di 10:1 salina Definty iniettando lentamente il volume richiesto di Definity in una siringa di soluzione fisiologica. Se una consegna infusione viene utilizzato, il Definity può essere ulteriormente diluita, a 50:1 o 100:1. </li><li> Capovolgere delicatamente la siringa per mescolare completamente l&#39;Definity e alle nebbie saline fino ad un aspetto ancora è raggiunto. Inversione della siringa immediatamente prima dell&#39;iniezione può essere necessaria anche se le bolle hanno cominciato a galleggiare di sospensione. </li><li> Calcolare il volume richiesto dosaggio in base a 0,02 ml / kg di Definity, o 0,2 mL / kg della soluzione (a 10:1 diluizione). </li></ol> 5. Ecografia di consegna <ol><li> Impostare i parametri sonicazione, utilizzando bassi scoppia duty cycle, non sonications onda continua. Sonicazione parametri adeguati a 0,5 MHz è 0,23 MPa di pressione situ, 10 ms scoppia a 1 Hz PRF, 2 min. sonicazione tempo totale. A 1,5 MHz appropriato in situ pressioni cadono attorno 0,45-0,5 MPa. </li></ol>_content &quot;&gt; pressioni appropriate a diverse frequenze può essere stimato utilizzando un indice di 0,46 meccanica 13. <ol start="2"><li> Verificare che la testa dell&#39;animale è ancora accoppiata all&#39;acqua. </li><li> Iniettare le microbolle lentamente nella vena della coda del catetere e lavare con 0,5 ml di soluzione salina normale. Iniziare la sonicazione contemporaneamente con l&#39;inizio dell&#39;iniezione. </li></ol> 6. Valutazione risonanza magnetica l&#39;esito del trattamento <ol><li> Dopo la sonicazione, iniettare un agente di contrasto MRI (ad esempio 0,2 ml / kg Omniscan, GE Healthcare) attraverso il catetere vena della coda seguita da un lavaggio 0,5 mL soluzione fisiologica. </li><li> Eseguire T 1-weighted imaging fino a quando il picco di contrasto è passato. Sonicazione siti che sono stati con successo perturbazione mostrerà miglioramenti più consistenti rispetto al tessuto circostante. </li><li> Eseguire T 2-weighted imaging per verificare edema. Segnale alto nei siti sonicazione è indicativo di edema. </li></ol> 7. RappresentanteRisultati Agenti di contrasto per risonanza magnetica può essere consegnato con successo attraverso la BBB con ultrasuoni focalizzati e diffusione microbolle. La figura 2 mostra pre e post-tipica FUS T 1-w immagini. Figura 2B mostra un maggiore contrasto (CE) T 1-w immagine con aperture focali distinte in quattro sedi sonicati. Sonicazione posizioni 1 e 2 mostrano enhancement particolarmente brillanti. In Fig.3 posizioni 1 e 2 può essere visto anche a corrispondere con T 2-w segnale alto, indicando edema. L&#39;estensione della T 2-w edema a volte può essere più facilmente visualizzati su fette sagittali. Figura 4 mostra CE-1-T e T w 2-w fette sagittali mediante sonicazione posizioni 1 e 3. L&#39;edema è visibile nella posizione 1, ma non posizione 3. Analisi spettrale dei dati acustici acquisiti sulle emissioni (Fig.5) possono mostrare le emissioni armoniche e / o delle emissioni armoniche sub / ultra quando cavitazione stabile è che si verificano. HarmonIC possono anche derivare da tessuto non-linearità, mentre le emissioni secondarie e ultraharmonic possono verificarsi come risultato di attività bolla 14. A pressioni più elevate emissioni a banda larga che indicano la cavitazione inerziale può anche essere rilevato. Tuttavia, questi sono stati associati con una maggiore quantità di stravasi eritrocitari e microlesioni che sonications senza cavitazione inerziale 11. L&#39;utilizzo dei risultati sonicazione frequenze più alte in aperture più localizzati a causa delle dimensioni più piccole macchia focale. Figura 6 mostra che alte frequenze può essere utilizzato per creare piccole regioni di apertura. Ciò consente analisi degli effetti a metà del cervello con un minor numero quasi cranio effetti. <img alt="Figura 1." src="/files/ftp_upload/3555/3555fig1.jpg" /> Figura 1. Impostazione sperimentale. <spuno class = &quot;pdflinebreak&quot;&gt; <img alt="Figura 2." src="/files/ftp_upload/3555/3555fig2.jpg" /> Figura 2. Pre (sinistra) e dopo (a destra) trattamento T 1-w immagini di cervello di ratto mostra enhancement in quattro sedi sonicazione. <img alt="Figura 3." src="/files/ftp_upload/3555/3555fig3.jpg" /> Figura 3. Pre (sinistra) e dopo (a destra) trattamento T 2-w immagini di cervello di ratto (come animale Fig.2) mostra T 2-w edema sonicazione in posizioni 1 e 2. <img alt="Figura 4." src="/files/ftp_upload/3555/3555fig4.jpg" /> Figura 4. Post trattamento sagittale T 1-w (sinistra) e T 2-w (destra) immagini dal cervello del ratto stesso come Figg. 2 e 3. L&#39;apertura nella posizione 1 (a sinistra) è correlata con T 2-w edema (a destra). Posizione 3 mostra edema di apertura (a sinistra), ma non T 2-w. </p&gt; <img alt="Figura 5." src="/files/ftp_upload/3555/3555fig5.jpg" /> Figura 5. Spettro delle frequenze a partire dai dati acquisiti nel corso di una singola raffica 10 ms a 551,5 kHz. La frequenza fondamentale (f 0) nonché armoniche (f 2 0) e sub / ultraharmonics (0,5 f 0, 1,5 f 0) sono visibili. <img alt="Figura 6." src="/files/ftp_upload/3555/3555fig6.jpg" /> Figura 6. Post trattamento CE-T 1-w assiale (a sinistra) e sagittale (a destra) le immagini da un cervello di ratto sonicato in quattro punti a 1,503 MHz. Aperture BBB a questa frequenza sono visti per essere più localizzata.

<ol>
	<li>Kinoshita, M., McDannold, N., Jolesz, F. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+localized+delivery+of+Herceptin+to+the+mouse+brain+by+MRI-guided+focused+ultrasound-induced+blood-brain+barrier+disruption.">Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11719-11723 (2006).</li><li>Jord&#227;o, J. F., Ayala-Grosso, C. A., Markham, K., Huang, Y., Chopra, R., McLaurin, J., Hynynen, K., Aubert, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+targeted+to+the+brain+with+image-guided+focused+ultrasound+reduces+amyloid-beta+plaque+load+in+the+TgCRND8+mouse+model+of+Alzheimer&#39;s+disease.">Antibodies targeted to the brain with image-guided focused ultrasound reduces amyloid-beta plaque load in the TgCRND8 mouse model of Alzheimer&#39;s disease.</a> PLoS One. 5 (5), e10549-e10549 (2010).</li><li>Liu, H. -. L., Hua, M. -. Y., Chen, P. -. Y., Chu, P. -. C., Pan, C. -. H., Yang, H. -. W., Huang, C. -. Y., Wang, J. -. J., Yen, T. -. C., Wei, K. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+with+focused+ultrasound+enhances+delivery+of+chemotherapeutic+drugs+for+glioblastoma+treatment.">Blood-brain barrier disruption with focused ultrasound enhances delivery of chemotherapeutic drugs for glioblastoma treatment.</a> Radiology. 255, 415-425 (2010).</li><li>Hynynen, K., McDannold, N., Vykhodtseva, N., Jolesz, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+MR+imaging-guided+focal+opening+of+the+blood-brain+barrier+in+rabbits.">Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.</a> Radiology. 220, 640-646 (2001).</li><li>Choi, J. J., Wang, S., Tung, Y. -. S., Morrison, B., Konofagou, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+of+various+pharmacologically-relevant+sizes+can+cross+the+ultrasound-induced+blood-brain+barrier+opening+in+vivo.">Molecules of various pharmacologically-relevant sizes can cross the ultrasound-induced blood-brain barrier opening in vivo.</a> Ultrasound Med. Biol. 36, 58-67 (2010).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+bottleneck+in+brain+drug+development.">The blood-brain barrier: bottleneck in brain drug development.</a> NeuroRx. 2, 3-14 (2005).</li><li>Bing, K. F., Howles, G. P., Qi, Y., Palmeri, M. L., Nightingale, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+&#40;BBB&#41;+disruption+using+a+diagnostic+ultrasound+scanner+and+Definity+in+mice.">Blood-brain barrier &#40;BBB&#41; disruption using a diagnostic ultrasound scanner and Definity in mice.</a> Ultrasound Med. Biol. 35, 1298-1308 (2009).</li><li>O'Reilly, M. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PVDF+receiver+for+ultrasound+monitoring+of+transcranial+focused+ultrasound+therapy.">A PVDF receiver for ultrasound monitoring of transcranial focused ultrasound therapy.</a> IEEE Trans. Biomed. Eng. 57, 2286-2294 (2010).</li><li>Chopra, R., Curiel, L., Staruch, R., Morrison, L., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+MRI-compatible+system+for+focused+ultrasound+experiments+in+small+animal+models.">An MRI-compatible system for focused ultrasound experiments in small animal models.</a> Med. Phys. 36, 1867-1874 (2009).</li><li>McDannold, N., Zhang, Y., Vykhodtseva, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Brain+Barrier+Disruption+and+Vascular+Damage+Induced+by+Ultrasound+Bursts+Combined+with+Microbubbles+can+be+Influenced+by+Choice+of+Anesthesia+Protocol.">Blood-Brain Barrier Disruption and Vascular Damage Induced by Ultrasound Bursts Combined with Microbubbles can be Influenced by Choice of Anesthesia Protocol.</a> Ultrasound Med. Biol. , (2011).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+blood-brain+barrier+with+focused+ultrasound:+association+with+cavitation+activity.">Targeted disruption of the blood-brain barrier with focused ultrasound: association with cavitation activity.</a> Phys. Med. Biol. 51, 793-807 (2006).</li><li>Yang, F. -. Y., Liu, S. -. H., Ho, F. -. M., Chang, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ultrasound+contrast+agent+dose+on+the+duration+of+focused-ultrasound-induced+blood-brain+barrier+disruption.">Effect of ultrasound contrast agent dose on the duration of focused-ultrasound-induced blood-brain barrier disruption.</a> J. Acoust. Soc. Am. 126, 3344-3349 (2009).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+induced+by+focused+ultrasound+and+circulating+preformed+microbubbles+appears+to+be+characterized+by+the+mechanical+index.">Blood-brain barrier disruption induced by focused ultrasound and circulating preformed microbubbles appears to be characterized by the mechanical index.</a> Ultrasound Med. Biol. 34, 834-840 (2008).</li><li>Neppiras, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+Cavitation.">Acoustic Cavitation.</a> Physics Reports (Review Section of Physics Letters). 61, 159-251 (1980).</li><li>O&#39;Reilly, M. A., Huang, Y., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+standing+wave+effects+on+transcranial+focused+ultrasound+disruption+of+the+blood-brain+barrier+in+a+rat+model.">The impact of standing wave effects on transcranial focused ultrasound disruption of the blood-brain barrier in a rat model.</a> Phys. Med. Biol. 55, 5251-5267 (2010).</li></ol>

K. Hynynen e R. Chopra sono co-fondatori del FUS Instruments, Inc. R. Chopra, A. e K. Waspe Hynynen sono azionisti in FUS Instruments, Inc. K. Hynynen riceve il sostegno alla ricerca dal FUS strumenti, Inc.

Preparazione degli animali e microbolle sono gli aspetti più critici di questa procedura. I capelli sulla testa dell&#39;animale deve essere completamente rimosso per evitare attenuazione del fascio di ultrasuoni. Il BBB può essere interrotto in isofluorano anestesia, tuttavia, diventa più difficile ottenere Dall&#39;apertura. Le microbolle devono sempre essere maneggiati con cura e di piccolo calibro, aghi di grande diametro deve essere utilizzato in sede di elaborazione, al fine di evit...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Piccoli Animali sistema a ultrasuoni focalizzati </td><td> FUS Instruments, Inc. </td><td> RK-100 </td></tr><tr><td> Definity </td><td> Lantheus Medical Imaging </td><td></td></tr><tr><td> Omniscan </td><td> GE Healthcare </td><td></td></tr></tbody></table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

Focused Ultrasound (FUS) a un&#39;interruzione della barriera emato-encefalica (BBB) ​​è una tecnica sempre più indagato per aggirare il 1-5 BBB. Il BBB è un ostacolo significativo per trattamenti farmacologici dei disturbi cerebrali quanto limita il passaggio delle molecole dalla vascolatura nel tessuto cerebrale di molecole meno di circa 500 Da dimensioni 6. FUS perturbazione indotta BBB (BBBD) è temporanea e reversibile 4 e ha un vantaggio rispetto chimica mezzo per indurre BBBD essendo altamente localizzata. FUS indotta BBBD fornisce un mezzo per verificare gli effetti di una vasta gamma di agenti terapeutici nel cervello, che non sarebbero altrimenti essere erogabili per il tessuto in concentrazione sufficiente. Mentre una vasta gamma di parametri ecografici si sono dimostrate efficaci a interrompere il BBB 2,5,7, ci sono diversi passaggi critici nella procedura sperimentale per garantire interruzioni di successo con la destinazione precisa. Questo protoccontorni olo come raggiungere MRI-guida FUS indotte BBBD in un modello murino, con un focus sulla preparazione degli animali critico e le fasi di manipolazione microbolle dell&#39;esperimento.

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Microbolle-mediata interruzione ultrasuoni focalizzati della barriera ematoencefalica (BBB) ​​è una tecnica promettente per non invasiva somministrazione di farmaci mirati nel cervello<sup&gt; 1-3</sup&gt;. Questo protocollo descrive la procedura sperimentale per la MRI-guidato interruzione transcranica BBB in un modello murino.

MRI-guidato Interruzione della barriera emato-encefalica transcranica con ultrasuoni focalizzati in un modello murino

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

mri-guided-disruption-blood-brain-barrier-using-transcranial-focused

Research

JoVE Journal

Medicine

17.8K Views.  Sunnybrook Research Institute. Microbolle-mediata interruzione ultrasuoni focalizzati della barriera ematoencefalica (BBB) ​​è una tecnica promettente per non invasiva somministrazione di farmaci mirati nel cervello 1-3. Questo protocollo descrive la procedura sperimentale per la MRI-guidato interruzione transcranica BBB in un modello murino.

Video: MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal Imaging di angiogenesi in un modello di ratto Nudo di metastasi ossee cancro al seno utilizzando la risonanza magnetica, tomografia computerizzata volumetrica e Ultrasuoni

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Ricerca

Medicina

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Modello Rat di emato-encefalica Turbativa Barriera per consentire mirato Therapeutics neurovascolare

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Un modello del Chronic Infusion nutrienti nel ratto

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Stretch in microvascolari cerebrali cellule endoteliali (CEND) come<em&gt; In Vitro</em&gt; Trauma cranico modello della barriera ematoencefalica

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

Misurare Crescente aortica Rigidità<em&gt; In Vivo</em&gt; In topi con ultrasuoni

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

L&#39;ossigeno-glucosio Deprivazione e riossigenazione come<em&gt; In Vitro</em&gt; Ischemia-riperfusione Injury modello per lo studio barriera sanguigna del cervello Erettile

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

MRI-guidata dmPFC-rTMS come Trattamento di trattamento resistente Disturbo Depressivo Maggiore

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

In vitro dosaggi per valutare la formazione e la rottura del vaso come la barriera emato-cerebrale

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

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) ...