This work is funded by the Cure for Cancer Foundation, Dutch Technology Foundation (STW) and The Netherlands Organisation for Health Research and Development (ZonMw).

La procedura presentata si svolge nell&#39;ambito di un protocollo di ricerca approvato dal Institutional Review Board della Academic Medical Center di Amsterdam, numero di registrazione NL41985.018. Il consenso informato scritto è richiesto da tutti i partecipanti. 1. Sistema <ol><li> Per questo esperimento, utilizzare un sistema di ottobre dominio Fourier, operante ad una banda di lunghezze d&#39;onda 1,280-1,350 nm 13. Dominio di Fourier bassa coerenza interferometria permette di scansione continua che aumenta la velocità di acquisizione dei dati rispetto ai sistemi ottobre dominio del tempo prima generazione. Nota: Il sistema PTOM interfacciato con una sonda a fibre ottiche, scansione elicoidale a ~ 90 °. Esso ha un diametro esterno di 2.7F (0,9 mm) e una lunghezza di 135 cm inseribile. La sonda si collega alla console ottobre attraverso un motore di azionamento e controllo ottico (dock di montaggio) con una gamma pullback di 54 mm. I set di dati acquisiti ottobre consistono in 541 immagini a sezione trasversale (B-scansioni) with una risoluzione assiale 15 micron (figura 1, 2). </li><li> Per garantire misure di attenuazione accurati e riproducibili, calibrazione misurando μ Oct per concentrazioni crescenti sulla base percentuale in peso di un&#39;emulsione grassa, (ad esempio, Intralipid) come descritto in precedenza da Kodach et al. 14, 15. In breve: <ol><li> Diluire un lotto standard emulsione lipidica 20% con demineralizzata H 2 O per ottenere concentrazioni di 0,125, 0,250, 0.5, 1.0, 2.0, 4.0, 10, 15 e 20 (azione) percento. <ol><li> Posizionare la sonda PTOM 200 ml di miscela di emulsione lipidica e acquisire una misurazione Office. </li><li> Cross reference estratto μ ottobre valori con i valori noti in letteratura. </li></ol></li></ol></li></ol> 2. Time Out e Posizionamento del paziente <ol><li> Prima di iniziare la procedura, eseguire un &quot;time out&quot; verifica nome, data di nascita, procedura, prlato ocedural, uso anticoagulante, e le allergie. </li><li> A seconda della posizione del tumore, posizionare il paziente sia in decubito prona o laterale. Fornire al paziente un sostegno adeguato e verificare se lui / lei aspetta di stare bene in questa posizione per un periodo da 20 a 40 min. </li><li> Utilizzando gli ultrasuoni (US) 16, localizzare il tumore e segnare il punto di entrata dell&#39;ago sulla pelle con inchiostro permanente. NOTA: Quando si usa la tomografia computerizzata (CT), utilizzare un modello di orientamento ago flessibile per localizzare la posizione preferita dell&#39;ago accesso. </li></ol> 3. Disinfezione e sterile Draping <ol><li> Mettere su un cappuccio del tappo e la bocca chirurgica. </li><li> Pulire la pelle intorno al sito di puntura con una soluzione di clorexidina / alcool, facendo attenzione a non rimuovere il segno di entrata dell&#39;ago precedentemente posizionato (punto 2.3). Disinfezione una vasta area impedirà la necessità di ulteriore pulizia in caso di imprevisto accesso dell&#39;ago riposizionamento. </li><li> Con reGard dei contenuti sterili, aprire il set percutanea puntura contenente: una siringa da 10 ml, un ago aspirato smussato, un G 21 ago per l&#39;iniezione, un bisturi, un 15 G coassiale introduttore ago, un G trocar ago 18, e 16 G nucleo biopsia pistola. </li><li> Lavarsi accuratamente le mani, applicando disinfettante per le mani dopo. Indossare un camice chirurgico e guanti sterili. </li><li> Coprire il paziente in teli sterili. </li><li> Applicare una copertura sterile intorno alla sonda ecografica e fissare la guida ago in posizione. </li></ol> 4. ottobre Preparazione <ol><li> Avviare la console ottobre e inserire i dati del paziente nei campi etichettati ID del paziente, cognome, nome e DOB (data di nascita) utilizzando l&#39;interfaccia della console. </li><li> Per quanto riguarda il contenuto sterili, decomprimere il pacchetto ottobre contenente una sonda ottobre, una copertura bacino di montaggio sterili, e una siringa luer-lock 5 ml. </li><li> Applicare la copertura sterile per il dock di montaggio console ottobre. Guida il dock di montaggio non sterile richiede laaiuto di un assistente. </li><li> Riempire la siringa da 5 ml con il 0,9% di NaCl e collegarlo alla porta di lavaggio. Lavare la sonda Ottobre finché appare acqua nella parte distale del coperchio sonda. </li><li> Caricare la sonda ottobre nel dock di montaggio. Dopo aver caricato la sonda ruoterà ed emettere luce rossa conferma corretto funzionamento. Lasciare la sonda nella copertura di protezione durante il lavaggio e caricamento per minimizzare il rischio di danni. </li><li> Rimuovere la sonda ottobre dalla sua copertina. Posizionare la sonda su una superficie dura e utilizzare un bisturi per accorciare la punta. Fissare la parte distale della sonda durante il taglio in modo da minimizzare la pressione sulla fibra ottica e prisma. Tagliare 5 millimetri distale dal prisma, utilizzando la (rossa) luce emessa per l&#39;orientamento. </li></ol> 5. Puncture <ol><li> Anestetizzare la pelle e strati profondi usando 2% lidocaina (20 mg / ml). Attendere alcuni minuti che consentono la lidocaina abbia effetto. Chiedere al paziente se non vi è alcun dolore. </li><li> Utilizzando la guida ago, posizionare il15 G coassiale introduttore ago verifica della posizione attraverso l&#39;imaging. Se il posizionamento è soddisfacente, rimuovere l&#39;otturatore (nucleo sharp ago). </li><li> Posizionare il 18 G trocar ago attraverso l&#39;ago introduttore, perforando il tumore. Ancora verificare la posizione dell&#39;ago con imaging. Se il posizionamento è soddisfacente rimuovere l&#39;otturatore. </li><li> Alimentare la sonda di ottobre l&#39;ago trocar fino sensazione di resistenza. </li><li> Mentre fissa la sonda ottobre, ritrarre l&#39;ago trocar, esponendo la sonda di Office per il tessuto tumorale. Mantenendo la punta dell&#39;ago trocar all&#39;interno del tumore minimizza attorcigliamento della sonda ottobre durante i cicli di respirazione. Questo riduce il rischio di danni sonda. </li><li> Ottobre Scan: <ol><li> Eseguire una scansione ottobre, con la console fissato a 541 B-scansioni al dataset. Il sistema qui utilizzato ottobre eseguirà un pullback automatico su una lunghezza di 5,4 centimetri richiedono alcuna regolazione di parametri specifici. </li><li> Controllare la scansione per qualità, manufatti e la comparsa di tessuto solido (Figura 1A). Artefatti più comunemente appaiono come bande circolari in piedi fuori dalla normale modello ottobre (Figura 1B). </li><li> Sostituire la sonda se artefatti persiste dopo ripetere la scansione. </li></ol></li><li> Ripetere il punto 5.6 fino ad un minimo di 3 set di dati OCT vengono acquisiti. </li><li> Rimuovere la sonda ottobre e trocar ago, lasciando l&#39;ago guida sul posto. </li><li> Braccio nucleo biopsia pistola e posizionarlo attraverso l&#39;ago introduttore, verificando la posizione sul imaging. </li><li> Se il posizionamento è soddisfacente, sparare la pistola biopsia. </li><li> Mettere materiale bioptico in un contenitore secondo il protocollo reparto di patologia. Qui, posto biopsie su una capsula di Petri con un intarsio di carta, sufficientemente sature di 0.9% NaCl. </li><li> Controllare la qualità nucleo biopsia e ripetere il punto 5.9 e 5.10 fino ad ottenere materiale sufficiente. </li></ol>

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The authors of this article have nothing to disclose.

In questa pubblicazione riportiamo sulla fattibilità di percutanea, ago base, ottobre del rene. Questo è un primo passo essenziale nello sviluppo di una tecnica PTOM nella clinicamente applicabile per differenziazione del tumore, definito come &quot;biopsia ottica&quot;. I nostri primi 25 pazienti hanno mostrato percutaneo ottobre di essere una procedura semplice e sicura. Una biopsia ottica ha due vantaggi rispetto biopsie convenzionali. In primo luogo, la vera acquisizione in tempo e l&#39;analisi dei dati ottobre f...

Gli ultimi decenni hanno mostrato un costante aumento dell&#39;incidenza di masse renali 1,2. Fino ad ora, le decisioni di trattamento di massa renali sono stati fatti principalmente sulla base di MRI e CT caratteristiche di imaging, età e comorbilità. Tuttavia questi metodi diagnostici e parametri clinici non hanno la finezza di rilevare veramente il potenziale maligno di una massa renale. Una biopsia core o agoaspirato con tessuto sufficiente per la valutazione patologica (diagnostica) fornisce differenziazione obiettiva del tumore sia con sensibilità e specificità nel range di 95-100% 3. Pertanto biopsia sta guadagnando accettazione nella valutazione delle masse renali sospette 4,5. Tuttavia, biopsie senza tessuto sufficiente a stabilire una diagnosi o con normale parenchima renale (non diagnostico) si verificano a un tasso del 10-20% nel complesso, e anche fino al 30% in piccole masse renali (&lt;4 cm MSR), ritardando il processo diagnostico per la necessità frequente per ulterioriprocedure di biopsia 3,5. Tomografia a coerenza ottica (OCT) è una modalità di imaging romanzo che ha il potenziale per superare gli ostacoli di cui sopra nella differenziazione massa renale. Sulla base della retrodiffusione della luce vicino infrarosso, OCT fornisce immagini con risoluzione assiale 15 micron ad una penetrazione nel tessuto effettiva di 2-3 mm (figura 1, 2). La perdita di intensità di segnale per millimetro di penetrazione del tessuto, una risultante di dispersione della luce tessuto-specifico, è espressa come coefficiente di attenuazione (μ Office: mm -1) come descritto da Faber et al 6.. Caratteristiche istologiche possono essere correlati a valori μ ottobre fornendo un parametro quantitativo di differenziazione tissutale (Figura 3). Durante la carcinogenesi, le cellule maligne mostrano un aumento del numero, nuclei più grandi e di forma irregolare con<html> un alto indice di rifrazione e mitocondri più attivo. A causa di questo sovraespressione di componenti cellulari, un cambiamento μ PTOM prevedibile quando si confrontano i tumori maligni tumori benigni o tessuto inalterato 7. Recentemente abbiamo studiato la capacità di superficiale ottobre distinguere tra masse renali benigne e maligne 8,9. In 16 pazienti, intra-operatorie misure ottobre del tessuto tumorale sono stati ottenuti utilizzando una sonda ottobre collocato esternamente. Il braccio di controllo costituito da misure ottobre di tessuto inalterato negli stessi pazienti. Tessuto normale ha mostrato un coefficiente di attenuazione mediana significativamente inferiore rispetto al tessuto maligno, confermando il potenziale dell&#39;OCT di differenziazione del tumore. Questa analisi quantitativa è stata applicata in modo simile a grade altri tipi di tessuto maligno, come carcinoma uroteliale 10,11 e vulvare differenziazione neoplasia epiteliale 12. ent &quot;&gt; Il nostro obiettivo è di sviluppare ottobre in una biopsia ottica, fornendo immagini in tempo reale in combinazione con il posto di differenziazione del tumore. L&#39;obiettivo del presente studio è quello di descrivere un percutanea, ago base, ottobre approccio nei pazienti con diagnosi di un massa renale valorizzazione solida. Questa descrizione metodo è, a nostra conoscenza, il primo a valutare la possibilità di ago basato ottobre di tumori renali.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>15G / 7.5cm Co-Axial Introducer Needle</td>
			<td>Angiotech, Gainesville, USA</td>
			<td>MCXS1612SX</td>
			<td></td>
		</tr>
		<tr>
			<td>18G / 20cm Trocar Needle</td>
			<td>Cook medical, Bloomington, USA</td>
			<td>DTN-18-20.0-U</td>
			<td></td>
		</tr>
		<tr>
			<td>16G / 20cm Quick-Core Biopsy Gun</td>
			<td>Cook Medical, Bloomington, USA</td>
			<td>G07827</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilumien Optis PCI Optimization System (OCT &#38; FFR)</td>
			<td>St. Jude medical, St. Paul, USA</td>
			<td>C408650</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Dragonfly Duo Imaging Catheter</td>
			<td>LightLab Imaging, Westford, USA</td>
			<td>C408644</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Sterile Dock Cover</td>
			<td>CFI Med. Solutions, Fenton, USA</td>
			<td>200-700-00</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>5ml Luer-lock Syringe</td>
			<td>Merit Med. Syst., South Jordan, USA</td>
			<td>C408647</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Blunt Fill Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>21G Injection Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>222434</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidoca&#239;ne HCl 2% (20mg/ml) solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>3624480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Gel, Aquasonic 100</td>
			<td>Parker Lab. Inc., Fairfield, USA</td>
			<td>GE424609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Cover</td>
			<td>Microtek Med., Alpharetta, USA</td>
			<td>PC1289EU</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA software package</td>
			<td colspan="2">FEI Visualization Sciences Group, Hillsboro, USA</td>
			<td>Software platform for 3D data analysis</td>
		</tr>
		<tr>
			<td>FIJI software package (open source)</td>
			<td>Open source, http://fiji.sc/Fiji</td>
			<td></td>
			<td>Open source image processing software</td>
		</tr>
	</tbody>
</table>

in vivo, percutaneous, needle based, optical coherence tomography of renal masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

Tra i primi 25 tumori (23 pazienti), sono stati eseguiti per un totale di 24 procedure ottobre successo. In un caso di malfunzionamento della sonda ha portato alla incapacità di acquisire una scansione Office. Due eventi avversi (AE) si è verificato, che sono descritte in dettaglio nella sezione di discussione. Caratteristiche generali paziente sono presentati nella tabella 1. La console ottobre è il software pre-installato che fornisce immagini di ottobre in tempo reale ...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

In Vivo, percutanea, Ago Based, Optical Coherence Tomography di masse renali

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

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In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

8.8K Views.  Academic Medical Center. Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Video: In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Doppler Optical Coherence Tomography di circolazione retinica

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

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Medicina

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

Near Infrared Tomografia ottica di proiezione per la valutazione delle β-cellule Grande Distribuzione in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Generazione e quantificazione 3-Dimensional di arteriosa lesioni nei topi mediante tomografia ottica di proiezione

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparazione e respirometrici valutazione dei mitocondri isolati dal tessuto muscolare scheletrico Ottenuto dalla biopsia percutanea

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

La valutazione transcutanea della funzione renale nei roditori Conscious

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

Tomografia ottica di coerenza di risoluzione ultraelevata Mouse per aiutare l'iniezione intraoculare nella ricerca retinica terapia genica

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

In Vivo Imaging dei topi Reporter Cx3cr1gfp/gfp con tomografia a coerenza ottica Spectral-domain e scansione Laser oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Valutazione della funzione del rene in modelli murini di malattia glomerulare

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...