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 procédure présentée se déroule dans un protocole de recherche approuvé par le Conseil de l&#39;Academic Medical Center d&#39;Amsterdam, numéro d&#39;enregistrement NL41985.018 Institutional Review. Le consentement éclairé écrit est exigé de tous les participants. 1. Système <ol><li> Pour cette expérience, utiliser un système OCT domaine de Fourier, fonctionnant à une longueur d&#39;onde 1,280-1,350 nm bande 13. domaine de Fourier interférométrie à faible cohérence permet un balayage continu qui augmente la vitesse d&#39;acquisition de données, par rapport aux systèmes dans le domaine temporel octobre première génération. Remarque: Le système est interfacé octobre avec une sonde à fibre optique, la numérisation en hélice à ~ 90 ° d&#39;angle. Il a un diamètre extérieur de 2.7f (0,9 mm) et une longueur insérable de 135 cm. La sonde se connecte à la console octobre par un moteur d&#39;entraînement et de commande optique (quai de montage) avec une gamme de retrait de 54 mm. Les ensembles de données acquises octobre se composent de 541 images en coupe transversale (B-scans) wvec une résolution axiale de 15 pm (figure 1, 2). </li><li> Afin de garantir des mesures précises et reproductibles atténuation, en mesurant étalonner octobre μ pour des concentrations en fonction du pourcentage en poids d&#39;une émulsion de matières grasses (par exemple, Intralipid) comme décrit précédemment par Kodach et al. 14, 15 augmente. En bref: <ol><li> Diluer un lot standard de 20% de matières grasses émulsion avec déminéralisée H 2 O pour obtenir des concentrations de 0,125, 0,250, 0,5, 1,0, 2,0, 4,0, 10, 15 et 20 (stock) pour cent. <ol><li> Placer la sonde octobre dans 200 ml de mélange d&#39;émulsion de graisse et d&#39;acquérir une mesure octobre. </li><li> Renvoi extrait μ octobre valeurs avec des valeurs connues dans la littérature. </li></ol></li></ol></li></ol> 2. Time Out et le positionnement des patients <ol><li> Avant de commencer la procédure, effectuer un &quot;time out&quot; vérifier le nom, date de naissance, la procédure, procedural côté, l&#39;utilisation anticoagulant, et les allergies. </li><li> Selon l&#39;emplacement de la tumeur, placer le patient en soit décubitus ventral ou latéral. Fournir au patient un soutien adéquat et vérifier se il / elle se attend à être à l&#39;aise dans cette position sur une période de 20 à 40 min. </li><li> Utilisation des ultrasons (US) 16, de localiser la tumeur et marquer le point sur ​​la peau avec de l&#39;encre permanente de pénétration de l&#39;aiguille. REMARQUE: Lorsque vous utilisez la tomodensitométrie (CT), utiliser un modèle d&#39;orientation de l&#39;aiguille souple pour localiser la position préférée de l&#39;aiguille d&#39;accès. </li></ol> 3. désinfection et stérile drapage <ol><li> Mettre un bonnet et la bouche couverture chirurgicale. </li><li> Nettoyer la peau autour du site de ponction en utilisant une solution de chlorhexidine / alcool, en prenant soin de ne pas supprimer l&#39;entrée de l&#39;aiguille marque déjà placé (étape 2.3). Désinfection une large zone empêche la nécessité d&#39;un nettoyage supplémentaire en cas d&#39;imprévu repositionnement de l&#39;aiguille d&#39;accès. </li><li> Avec regard du contenu stérile, ouvrir l&#39;ensemble percutanée à la perforation comprenant: une seringue de 10 ml, une aiguille d&#39;aspiration émoussée, une aiguille de calibre 21 d&#39;injection, un scalpel, un co-axial aiguille d&#39;introduction 15 de G, G aiguille 18 de trocart, et une 16 G biopsie pistolet. </li><li> Se laver les mains, appliquer désinfectant pour les mains par la suite. Mettez sur une robe chirurgical et des gants stériles. </li><li> Couvrir le patient dans des champs stériles. </li><li> Appliquer une couverture stérile autour de la sonde à ultrasons et fixer le guide de l&#39;aiguille en place. </li></ol> 4. Préparation octobre <ol><li> Démarrez la console PTOM et entrer dans les détails du patient dans les champs marqués identification du patient, le nom, prénom et date de naissance (date de naissance) en utilisant l&#39;interface de la console. </li><li> En ce qui concerne le contenu stérile, déballer le paquet contenant une sonde octobre octobre, une couverture stérile quai de montage, et un 5 ml luer-lock seringue. </li><li> Appliquer le couvercle stérile pour le quai de montage console octobre. Guider le quai de montage non stérile exige laaide d&#39;un assistant. </li><li> Remplir la seringue de 5 ml NaCl à 0,9% et l&#39;attacher au port de rinçage. Rincer la sonde octobre que l&#39;eau apparaît dans la partie distale du couvercle de la sonde. </li><li> Chargez la sonde octobre dans la station de montage. Après le chargement de la sonde va tourner et émettre de la lumière rouge confirmant bon fonctionnement. Laisser la sonde dans sa housse de protection pendant le rinçage et le chargement de minimiser le risque de dommages. </li><li> Retirer la sonde du PTOM de sa couverture. Placer la sonde sur une surface dure et d&#39;utiliser un scalpel pour raccourcir la pointe. Fixer la partie distale de la sonde pendant la coupe afin de minimiser la pression sur la fibre optique et le prisme. Coupez 5 mm distale du prisme, en utilisant le (rouge) la lumière émise pour l&#39;orientation. </li></ol> 5. Ponction <ol><li> Anesthésier la peau et les couches profondes à l&#39;aide de lidocaïne à 2% (20 mg / ml). Attendez quelques minutes permettant la lidocaïne pour prendre effet. Demandez au patient se il est une douleur. </li><li> Utilisation du guide d&#39;aiguille, placez le15 G co-axiale aiguille d&#39;introduction vérifier la position grâce à l&#39;imagerie. Si le placement est satisfaisante, retirez l&#39;obturateur (de noyau d&#39;aiguille dièse). </li><li> Placez le G aiguille de trocart 18 à travers l&#39;aiguille d&#39;introduction, perçant la tumeur. Encore une fois vérifier la position de l&#39;aiguille à l&#39;imagerie. Si le placement est satisfaisante retirer l&#39;obturateur. </li><li> Nourrir la sonde octobre l&#39;aiguille de trocart jusqu&#39;à sentir une résistance. </li><li> Tout en fixant la sonde octobre, rétracter l&#39;aiguille de trocart, exposant la sonde PTOM dans le tissu tumoral. Garder la pointe de l&#39;aiguille de trocart dans la tumeur minimise le vrillage de la sonde octobre pendant les cycles de respiration. Cela réduit le risque de dommages de la sonde. </li><li> Balayage OCT: <ol><li> Effectuer une analyse octobre, avec la console fixé à 541 B-scans par ensemble de données. Le système utilisé ici octobre effectuera un retrait automatique sur une longueur de 5,4 cm nécessitant aucun réglage de paramètres spécifiques. </li><li> Consultez l&#39;analyse de la qualité, des artefacts et l&#39;apparition de tissu solide (Figure 1A). Artefacts apparaissent le plus souvent sous forme de bandes circulaires se démarquer de la tendance normale octobre (figure 1B). </li><li> Remplacer la sonde si artefacts persiste après une nouvelle analyse. </li></ol></li><li> Répétez l&#39;étape 5.6 jusqu&#39;à un minimum de 3 ensembles de données PTOM sont acquises. </li><li> Retirer la sonde PTOM et l&#39;aiguille de trocart, laissant l&#39;aiguille d&#39;introduction en place. </li><li> Armez le canon biopsie et placez-le à travers l&#39;aiguille d&#39;introduction, la vérification de la position sur l&#39;imagerie. </li><li> Si le positionnement est satisfaisant, tirer le pistolet à biopsie. </li><li> Placez le matériel de biopsie dans le récipient selon le protocole de service de pathologie. Ici, place des biopsies sur une boîte de Pétri avec une incrustation de papier, suffisamment saturés de NaCl à 0,9%. </li><li> Vérifiez la qualité de biopsie de base et répétez l&#39;étape 5.9 et 5.10 jusqu&#39;à suffisamment de matériau est obtenu. </li></ol>

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The authors of this article have nothing to disclose.

Dans cette publication, nous présentons sur la faisabilité d&#39;percutanée, aiguille base, octobre du rein. Ce est une première étape essentielle dans le développement de l&#39;OCT dans une technique applicable cliniquement pour la différenciation de la tumeur, appelé comme un «biopsie optique». Nos premiers 25 patients ont montré percutanée octobre pour être une procédure simple et sûre. Une biopsie optique a deux avantages par rapport aux biopsies de base classiques. Tout d&#39;abord, l&#39;acquisition...

Les dernières décennies ont montré une augmentation constante de l&#39;incidence de 1,2 masses rénales. Jusqu&#39;à présent, les décisions de traitement de masse rénales ont été réalisées principalement sur la base des caractéristiques d&#39;imagerie IRM et de TDM, l&#39;âge et la comorbidité. Cependant, ces méthodes de diagnostic et les paramètres cliniques ne ont pas la finesse de détecter vraiment le potentiel de malignité d&#39;une masse rénale. Une biopsie au trocart ou l&#39;aspiration à l&#39;aiguille fine avec le tissu suffisant pour une évaluation pathologique (diagnostic) fournit la différenciation tumorale objective à la fois la sensibilité et la spécificité de l&#39;ordre de 95 à 100% 3. Par conséquent biopsie est plus accepté dans l&#39;évaluation des masses rénales suspectes 4,5. Cependant, les biopsies sans suffisamment de tissu pour établir un diagnostic ou normale parenchyme rénal (non-diagnostic) se produisent à un taux de 10 à 20% dans l&#39;ensemble, et même jusqu&#39;à 30% dans les petites masses rénales (&lt;4 cm, MRS), retardant le processus de diagnostic en raison de la nécessité fréquente pour supplémentairesprocédures de biopsie 3,5. Tomographie par cohérence optique (OCT) est une nouvelle technique d&#39;imagerie qui a le potentiel de surmonter les obstacles mentionnés ci-dessus dans la différenciation de la masse rénale. Sur la base de la rétrodiffusion de la lumière proche infrarouge, octobre fournit des images ayant une résolution axiale de 15 um à une pénétration tissulaire effective de 3.2 mm (figure 1, 2). La perte d&#39;intensité de signal par millimètre de pénétration tissulaire, résultant d&#39;une diffusion de lumière spécifique d&#39;un tissu, est exprimée par le coefficient d&#39;atténuation (μ octobre: ​​-1 mm) comme décrit par Faber et al 6.. Caractéristiques histologiques peuvent être corrélées à des valeurs μ octobre fournissant un paramètre quantitatif pour la différenciation de tissu (figure 3). Au cours de la cancérogenèse, les cellules malignes présentent un plus grand nombre, des noyaux de plus en plus en forme irrégulière avec<html> un indice de réfraction plus élevé et des mitochondries plus active. En raison de cette surexpression de composants cellulaires, un changement de μ octobre est à prévoir lors de la comparaison à des tumeurs malignes ou des tumeurs bénignes du tissu affecté 7. Récemment, nous avons étudié la capacité des superficielle octobre de différencier entre les masses rénales bénignes et malignes 8,9. Chez 16 patients, les mesures octobre intra-opératoires de tissu tumoral ont été obtenus en utilisant une sonde octobre placé à l&#39;extérieur. Le bras de commande composé de mesures octobre de tissu affecté dans les mêmes patients. Tissus normaux a montré un coefficient médian significativement plus faible d&#39;atténuation par rapport à un tissu malin, confirmant le potentiel de l&#39;OCT de différenciation de la tumeur. Cette analyse quantitative a été appliqué d&#39;une manière similaire à d&#39;autres types de qualité tissu malin, carcinome urothélial tels que 10,11 et la différenciation de la néoplasie épithéliale vulvaire 12. ent &quot;&gt; nous visons à développer octobre dans une biopsie optique, fournissant l&#39;imagerie en temps réel combinée avec la différenciation de la tumeur sur le terrain. L&#39;objectif de la présente étude est de décrire un percutanée, aiguille base, octobre approche chez les patients diagnostiqués avec un Amélioration de la masse rénale solide. Cette méthode description est, à notre connaissance, le premier à évaluer la possibilité de l&#39;aiguille octobre base des tumeurs rénales.

<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.

Parmi les 25 premières tumeurs (23 patients), un total de 24 procédures octobre réussies ont été effectuées. Dans un cas, un dysfonctionnement de la sonde conduit à l&#39;incapacité d&#39;acquérir une analyse PTOM. Deux événements indésirables (EI) a eu lieu, qui sont décrits en détail dans la section de discussion. Caractéristiques générales des patients sont présentées dans le tableau 1. La console octobre a logiciels pré-installés fournissant en temps...

Watch this Scientific Journal Video about In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses at JoVE.com

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, percutanée, Aiguille Based, tomographie par cohérence optique des masses rénales

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

in-vivo-percutaneous-needle-based-optical-coherence-tomography-renal

Research

JoVE Journal

Medicine

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

8.8K vues.  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.

Vidéo: 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.

Tomographie par cohérence optique Doppler de la circulation rétinienne

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

Recherche

Médecine

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.

Près de tomographie optique de projection infrarouge pour l&#39;évaluation de la distribution de masse β-cellulaire en recherche sur le diabète

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.

Génération et quantification 3-Dimensional des lésions artérielles chez la souris utilisant la tomographie optique de projection

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 L&#39;imagerie optique de tumeurs cérébrales et l&#39;arthrite Utilisation fluorescent SAPC-DOPS nanovésicules

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.

Préparation et respirométrique évaluation des mitochondries isolées de tissu musculaire squelettique Obtenu par biopsie percutanée aiguille

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.

Évaluation transcutanée de la fonction rénale dans Rongeurs 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.

Appauvrissement des cellules de souris de xénogreffes de tumeurs humaines améliore de manière significative l&#39;analyse en aval des cellules cibles

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.

Tomographie à cohérence optique souris ultra haute résolution pour faciliter l’Injection intraoculaire dans la recherche de la thérapie génique rétinienne

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 Imagerie des souris journaliste Cx3cr1gfp/gfp avec domaine Spectral Optical Coherence Tomography et numérisation Laser ophtalmologique

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.

Évaluation de la fonction rénale chez les modèles murins de la maladie glomérulaire

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