This work was supported by the National Institutes of Health (R01-EB014496). The authors would like to thank Iason Apostolakis for his contribution to the experiments.

Questo protocollo è stato approvato dalla cura e l&#39;uso degli animali Comitato Istituzionale della Columbia University. Tutto l&#39;acquisizione e l&#39;elaborazione dei dati sono state eseguite utilizzando l&#39;ambiente Matlab. 1. set-up sperimentale <ol><li> Degassare un vivo canino campione ex fegato durante 90 min. Posizionare il campione di fegato in una vasca riempita con tampone fosfato degasato Solution (Figura 1). Fissare il campione di fegato su un assorbitore acustico con aghi alle estremità del fegato. </li><li> Inserire un 64-elemento, mm di passo 0,32, 2,5 MHz frequenza centrale phased array (imaging) attraverso un foro circolare situato al centro di un 93-elemento dell&#39;array emisferica HIFU trasduttore (terapeutico) a 4,5 MHz frequenza centrale, 70 mm profondità focale e 1,7 mm x 0,4 millimetri di dimensione focale 11. Allineare i due trasduttori coassialmente e fissare il trasduttore di imaging nella trasduttore terapeutica con viti di regolazione. <ol><li> Coprire il trasduttore HIFU con avolume controllato membrana poliuretanica riempita con acqua corrente degasata per raffreddarlo. Montare il trasduttore su un 3-D posizionatore controllati dal computer. </li></ol></li><li> Collegare il trasduttore HIFU ad un generatore di funzione di invio a 25 Hz modulata in ampiezza della forma d&#39;onda sinusoidale con 500 mV massima ampiezza. Collegare il trasduttore di imaging per un sistema ad ultrasuoni completamente programmabile utilizzando il software Matlab. Nota: Un software associato con il sistema ad ultrasuoni e con l&#39;ambiente Matlab deve essere installato sul computer collegato al sistema. Un amplificatore 50 dB RF e una rete di adattamento deve essere collocato tra il trasduttore HIFU e il generatore di funzioni per amplificare rispettivamente la potenza e abbinare l&#39;impedenza. </li><li> Creare una griglia polare, utilizzando Matlab, iniziando 50 mm dalla superficie della matrice e 40 mm di profondità in direzione radiale con passo spaziale di 9,625 micrometri e di 90 ° in direzione azimutale con 128 linee e la cui origine è il focus dell&#39;onda divergenti. Definire la sorgente del divergenti dell&#39;onda 10.24 mm (metà delle dimensioni dell&#39;apertura) dietro la superficie della matrice e centrato nella direzione laterale. <ol><li> Creare una griglia cartesiana, utilizzando Matlab, iniziando 50 mm dalla superficie della matrice e 40 mm di profondità in senso assiale con passo spaziale di 9,625 micron e 20 mm di larghezza in direzione laterale con 64 linee per l&#39;onda piana. Definire la sorgente dell&#39;onda piana sulla superficie della matrice. Per ogni griglia, calcolare il tempo dalla sorgente a ciascun punto della griglia e di nuovo a ogni elemento dell&#39;array. </li></ol></li><li> Inserire &quot;ReconMat_DW&quot; per l&#39;imaging divergenti onda o &quot;ReconMat_PW&quot; per l&#39;imaging onda piana nella finestra di comando Matlab e premere il tasto &quot;Enter&quot; per creare una matrice di ricostruzione associato con un algoritmo di ritardo-e-somma standard per ogni griglia. Applicare l&#39;algoritmo di ritardo-e-somma ogni vettore di base tipo e recuperare i elemen non zerits della matrice risultante 11. Assegnare gli elementi non nulli ottenuti dalla matrice risultante alla matrice sparse nella posizione corrispondente. Salvare la matrice di ricostruzione sul disco rigido del computer. Nota: Il divergenti e metodi d&#39;onda piano utilizzano due matrici distinte di ricostruzione. <ol><li> Gettate la matrice di ricostruzione di una matrice GPU. Inserire &quot;SetUpP4_2Flash_4B_streaming_DW&quot; per divergenze di imaging onda o &quot;SetUpP4_2Flash_4B_streaming_PW&quot; per l&#39;imaging onda piana nella finestra di comando Matlab e premere &quot;Invio&quot; per creare un file di installazione per l&#39;acquisizione dei dati del canale ultrasuoni utilizzando lo script associato al phased array e fornito dal produttore del sistema ad ultrasuoni. Nome del file di setup &quot;P4-2Flash_DivergingWave.mat&quot; per l&#39;imaging divergenti onda e &quot;P4-2Flash_PlaneWave.mat&quot; per l&#39;imaging onda piana. Nota: Un pacchetto software commerciale deve essere installato sul computer to lanciare la matrice sparsa di ricostruzione di una matrice GPU. </li></ol></li><li> Sincronizzare il sistema a ultrasuoni con il generatore di funzione utilizzando un trigger esterno in modo che l&#39;acquisizione dei dati frame rate elevato ultrasuoni del fegato inizia contemporaneamente come HIFU. </li><li> Aprire Matlab. Eseguire lo script di installazione &quot;SetUpP4_2Flash_4B.m&quot; fornito dal produttore del sistema ad ultrasuoni di utilizzare immagini B-mode. Nome del file di installazione creato: &quot;P4-2Flash_4B_Bmode.mat&quot;. Utilizzare il comando &quot;VSX&quot; e quando &quot;Nome del file di .mat a processo:&quot; viene richiesto, inserire il nome del file di setup &quot;P4-2Flash_4B_Bmode.mat&quot;. Spostare entrambi i trasduttori e utilizzare il display B-mode che è apparso sullo schermo del computer per posizionarli nella regione di destinazione del fegato per l&#39;ablazione. Target una regione di circa 1 cm sotto la superficie del fegato per evitare un&#39;elevata attenuazione ultrasuoni causa dell&#39;assorbimento. Salvare un&#39;immagine convenzionale B-mode del fegato nel computer. Nota:Qui ci siamo esibiti ablazioni HIFU a 11 luoghi diversi in due esemplari di fegato spostando i trasduttori con il posizionatore 3-D per ogni ablazione. </li></ol> 2. Ecografia Acquisizione Dati <ol><li> Aprire Matlab. Utilizzare il comando &quot;VSX&quot; e quando &quot;Nome del file di .mat a processo:&quot; viene richiesto, immettere il nome del file di setup &quot;P4-2Flash_DivergingWave.mat&quot; per l&#39;imaging divergenti onda o &quot;P4-2Flash_PlaneWave.mat&quot; per onda piana imaging. Avviare l&#39;HIFU e applicarlo durante 2min alla regione di destinazione. </li><li> Acquisire i dati del canale RF a 1.000 fotogrammi al secondo durante 2 minuti utilizzando onde divergenti. In alternativa, acquisire i dati del canale RF a 1.000 fotogrammi al secondo durante 2 min con onde piane. </li><li> Trasferire i dati a un computer host ogni 200 fotogrammi con un cavo PCI Express. In alternativa, per streaming in tempo reale, acquisire i dati del canale RF in 167 fotogrammi al secondo durante 2 minuti utilizzando onde piane e trasfer i dati a un computer host ogni 2 fotogrammi. Nota: I metodi di imaging con l&#39;insieme delle 200 fotogrammi fornisce ad alta risoluzione temporale all&#39;interno di ogni set, ma la creazione di spazi vuoti tra ogni serie ed è appropriato per l&#39;elaborazione off-line. Il metodo di imaging a 167 fps con una risoluzione temporale inferiore, ma non crea eventuali lacune in tutto il tutto il tempo di ablazione ed è adatto per streaming in tempo reale. </li><li> Gettate la matrice di dati canale RF per una sola matrice precisione GPU con Matlab. Moltiplicare la matrice di dati canale RF dalla matrice di ricostruzione di ottenere i dati RF ricostruiti 11. </li></ol> 3. Spostamento Imaging <ol><li> Creare un 6 ° ordine Butterworth filtro passa-basso a 4 MHz frequenza di taglio utilizzando il sistema DSP System Toolbox di Matlab. Applicare questo filtro passa-basso per i dati RF ricostruiti per filtrare la componente 4,5 MHz HIFU. </li><li> Stimare lo spostamento assiale tra fotogrammi consecutivi utilizzando 1-D normalizzato cross-correlazionecon una lunghezza 3,1 millimetri-finestra e il 90% di sovrapposizione. </li><li> Creare un 6 ° ordine Butterworth filtro passa-basso a 100 Hz frequenza di taglio utilizzando il sistema DSP System Toolbox di Matlab. Applicare questo filtro passa basso per i dati di spostamento temporale utilizzando Matlab per recuperare la componente di frequenza 50 Hz-oscillatorio. </li><li> Definire una regione di interesse (ROI) come la regione focale a -6 dB (1,7 x 0,4 mm in acqua) e si trova a distanza di 70 mm dalla superficie del trasduttore. Estrarre i dati di spostamento in questo ROI. Stima il rapporto segnale-rumore rapporto di spostamento (SNR d) in corrispondenza della zona focale dopo 2 min di ablazione come il rapporto fra il volume media e la deviazione standard dello spostamento nella ROI. </li><li> Estrarre il segnale di spostamento temporale di 50 Hz al fuoco dai dati della matrice di spostamento. Convertire il segnale di spostamento temporale al fuoco in suoni udibili con Matlab. </li></ol>

<ol>
	<li>Al-Bataineh, O., Jenne, J., Huber, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+future+applications+of+high+intensity+focused+ultrasound+in+cancer.">Clinical and future applications of high intensity focused ultrasound in cancer.</a> Cancer Treat Rev. 38, 346-353 (2012).</li><li>Dewhirst, M. W., Viglianti, B. L., Lora-Michiels, M., Hanson, M., Hoopes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+principles+of+thermal+dosimetry+and+thermal+thresholds+for+tissue+damage+from+hyperthermia.">Basic principles of thermal dosimetry and thermal thresholds for tissue damage from hyperthermia.</a> Int J Hyperthermia. 19, 267-294 (2003).</li><li>Napoli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR-guided+high-intensity+focused+ultrasound:+current+status+of+an+emerging+technology.">MR-guided high-intensity focused ultrasound: current status of an emerging technology.</a> Cardiovasc Intervent Radiol. 36, 1190-1203 (2013).</li><li>Gudur, M. S., Kumon, R. E., Zhou, Y., Deng, C. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+rapid+B-mode+ultrasound+imaging+for+real-time+monitoring+of+lesion+formation+and+gas+body+activity+during+high-intensity+focused+ultrasound+ablation.">High-frequency rapid B-mode ultrasound imaging for real-time monitoring of lesion formation and gas body activity during high-intensity focused ultrasound ablation.</a> IEEE Trans Ultrason Ferroelectr Freq Control. 59, 1687-1699 (2012).</li><li>Jensen, C. R., Cleveland, R. O., Coussios, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+temperature+estimation+and+monitoring+of+HIFU+ablation+through+a+combined+modeling+and+passive+acoustic+mapping+approach.">Real-time temperature estimation and monitoring of HIFU ablation through a combined modeling and passive acoustic mapping approach.</a> Phys Med Biol. 58, 5833-5850 (2013).</li><li>Mariani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+shear+waves+elastography+monitoring+of+thermal+ablation:+in+vivo+evaluation+in+pig+livers.">Real time shear waves elastography monitoring of thermal ablation: in vivo evaluation in pig livers.</a> J Surg Res. 188, 37-43 (2014).</li><li>Bing, K. F., Rouze, N. C., Palmeri, M. L., Rotemberg, V. M., 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=Combined+ultrasonic+thermal+ablation+with+interleaved+ARFI+image+monitoring+using+a+single+diagnostic+curvilinear+array:+a+feasibility+study.">Combined ultrasonic thermal ablation with interleaved ARFI image monitoring using a single diagnostic curvilinear array: a feasibility study.</a> Ultrason Imaging. 33, 217-232 (2011).</li><li>Athanasiou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+lesions:+quantitative+elastography+with+supersonic+shear+imaging--preliminary+results.,+Radiology.">Breast lesions: quantitative elastography with supersonic shear imaging--preliminary results., Radiology.</a> 256, 297-303 (2010).</li><li>Maleke, C., Konofagou, E. 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The authors declare that they have no competing financial interests.

Monitoraggio in tempo reale delle lesioni HIFU è importante per garantire una corretta ed efficiente consegna lesione. Poiché le forme di lesione, il tessuto si irrigidisce e la sua ampiezza di movimento sotto eccitazione diminuisce. Applicando HIFU in una regione dei risultati di tessuto in una forza di radiazione acustica che induce lo spostamento del tessuto. La variazione relativa di spostamento è un surrogato di variazione relativa di rigidità dei tessuti. Questa tecnica offre il vantaggio di monitorare HIFU le...

High Intensity Focused Ultrasound (HIFU) is a technique that generates temperature elevation at the focal region and can be used to ablate cancerous tissue 1. Temperature elevation at the focus causes thermal lesions in the tissue 2. In order to avoid overtreating a region and to reduce treatment duration, it is imperative to reliably monitor the ablation. Magnetic resonance-guided focused ultrasound (MRgFUS) is the main technique used in clinic to guide and monitor HIFU treatment 3. MRI provides high spatial resolution images of the treated region with tissue displacement or thermal dose but has a frame rate of 0.1-1 Hz and is costly. Several ultrasound-based techniques such as B-mode imaging 4, passive acoustic mapping 5, shear wave imaging 6 and acoustic radiation force impulse 7 have been developed to guide and monitor thermal ablation. However, B-mode imaging and passive acoustic mapping do not provide imaging of mechanical properties of the ablated region which is useful to the operator to improve lesion delivery. 
Shear wave imaging and acoustic radiation force impulse can both characterize the elasticity of the tissue by measuring acoustic radiation force-induced displacements 7,8. However, in both methods, the HIFU treatment is typically interrupted to monitor the ablation. Our group has developed a technique called Harmonic Motion Imaging for Focus Ultrasound (HMIFU) which can monitor the HIFU treatment with ultrasound without stopping the ablation9,10. Briefly, a HIFU transducer sends an amplitude-modulated wave to the region to ablate while simultaneously generating an oscillatory motion in the focal region. A co-axially aligned ultrasound transducer is used to image this oscillation. The magnitude of the induced motion is related to the stiffness of the tissue.
To ensure proper lesion delivery, the temporal resolution of real-time monitoring is of key interest in ablation guidance. Recently, our group has shown real-time streaming of displacement at a frame rate up to 15 Hz, imaged with diverging waves in a narrow field of view and using a fast image reconstruction method 11. Several beamforming techniques can be used to image the displacements. A large field of view can be obtained with diverging wave imaging by changing the delay profile but the axial direction is not aligned with the HIFU beam on the lateral regions and the wave is attenuated due to geometric spreading in the lateral direction, which can affect the quality of the displacement estimation. In contrast, the lateral field of view for plane wave is upper bounded by the active aperture but the axial direction is aligned with the HIFU beam at the focus and there is no geometric spreading in the lateral direction. Depending on the type of application, one or the other imaging method can be selected. The objectives of this protocol are to show how plane wave imaging can provide real-time streaming of displacements images using HMIFU during ablation and to compare the quality of the motion estimation between diverging and plane wave imaging.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>P4-2 Phased array</td>
			<td>ATL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H-178 HIFU transducer</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-D positioner</td>
			<td>Velmex Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AT33522A function generator</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 ultrasound system</td>
			<td>Verasonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3100L RF amplifier</td>
			<td>ENI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matching network</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Degasing system</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Programming software</td>
			<td>Matlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jacket software package</td>
			<td>Accelereyes</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time monitoring of high intensity focused ultrasound (hifu) ablation of in vitro canine livers using harmonic motion imaging for focused ultrasound (hmifu)

Harmonic Imaging Movimento per Focused Ultrasound (HMIFU) è una tecnica in grado di eseguire e monitorare High Intensity Focused Ultrasound (HIFU) ablazione. Un moto oscillatorio viene generato al centro di un 93-elemento e 4,5 MHz frequenza centrale HIFU trasduttore applicando un segnale modulato in ampiezza 25 Hz utilizzando un generatore di funzione. A 64-elemento e 2,5 MHz trasduttore di imaging con 68kPa pressione di picco è confocally posto al centro del trasduttore HIFU per acquisire i dati del canale a radiofrequenza (RF). In questo protocollo, il monitoraggio in tempo reale della termoablazione con HIFU con una potenza acustica di 7 W in fegati canino in vitro è descritta. Trattamento HIFU è applicato sul tessuto durante 2 minuti e la regione ablato è ripreso in tempo reale utilizzando l&#39;imaging divergenti o un&#39;onda piana fino a 1.000 fotogrammi / secondo. La matrice dei dati del canale RF viene moltiplicata per una matrice sparsa per la ricostruzione dell&#39;immagine. Il campo ricostruito di vista è di 90 ° per divergente wave e 20 mm per l&#39;imaging onde piane ed i dati sono campionati a 80 MHz. La ricostruzione viene eseguita su una Processing Unit grafica (GPU) per immagini in tempo reale ad un frame rate 4.5 display. 1-D normalizzato cross-correlazione dei dati RF ricostruito viene utilizzato per stimare spostamenti assiali nella regione focale. L&#39;entità dello spostamento picco-picco alla profondità focale diminuisce durante l&#39;ablazione termica che denota irrigidimento del tessuto a causa della formazione di una lesione. Il segnale-rumore rapporto di spostamento (SNR d) presso l&#39;area focale di un&#39;onda piana è stata di 1,4 volte superiore a quello divergente onda mostrare che l&#39;imaging onda piana sembra produrre meglio spostamento mappe di qualità per HMIFU di divergenza di imaging onda.

In tempo reale lo streaming di operatore spostamento durante HIFU l&#39;ablazione può essere ottenuto utilizzando l&#39;imaging onda divergenti e aereo. Figura 2 è un cattura schermo video che mostra visualizzazione in tempo reale della forza radiazioni acustico spostamento indotto con piano di imaging onda in vitro cane fegati in corso HIFU l&#39;ablazione . Gli spostamenti sono in streaming in tempo reale sullo schermo del computer con un frame rate di visualizzazione di 4.5 Hz. Sp...

Watch this Scientific Journal Video about Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU a

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoraggio in tempo reale di alta intensità ultrasuoni focalizzati (HIFU) Ablazione<em&gt; In Vitro</em&gt; Canine Livers Uso Harmonic Imaging Movimento per Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

real-time-monitoring-high-intensity-focused-ultrasound-hifu-ablation

Research

JoVE Journal

Bioengineering

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

9.9K Views.  Columbia University. This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Video: Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

Mezzo di contrasto ecografico mirato trattamento dei gliomi nei topi tramite Drug-Bearing consegna delle nanoparticelle e ablazione microvascolari

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Ricerca

Bioingegneria

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Decellularization e Recellularization di fegati interi

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

L&#39;induzione di segnali di adesione-dipendenti utilizzando ultrasuoni a bassa intensità

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Wideband rivelatore ottico di ultrasuono per applicazioni Medical Imaging

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Universale Mano libera tridimensionale optoacustica Imaging Probe per Deep Tissue angiografia umana e funzionali studi preclinici in tempo reale

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Oculare Test (EIT) per identificazione del pericolo di sostanze chimiche irritanti oculari utilizzando Ricostruito umana Cornea come epiteliale (RHCE) Tessuto Modello

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

Monitoraggio in tempo reale di intracellulare Bile Acid Dynamics Utilizzando una sede a FRET geneticamente codificato Bile Acid sensore

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

Corretto posizionamento e trattenuta di un ratto dell'arto per l'Imaging mirato ad alta risoluzione dell'osso utilizzando Micro-architettura In Vivo Micro-tomografia computerizzata

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Mano clinico fotoacustico sistema di Imaging per Imaging animale piccolo Non-invasivo in tempo reale

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

Focalizzata la litografia del fascio ionico per esacire di nano-architetture nei microelettrodi

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...