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

El procedimiento presentado se lleva a cabo bajo un protocolo de investigación aprobado por la Junta de Revisión Institucional del Centro Médico Académico de Amsterdam, número de registro NL41985.018. Consentimiento informado por escrito se requiere de todos los participantes. 1. Sistema <ol><li> Para este experimento, utilizar un sistema OCT de dominio Fourier, que opera a una banda de longitud de onda de 1,280-1,350 nm 13. Dominio de Fourier interferometría de baja coherencia permite la exploración continua que aumenta la velocidad de adquisición de datos en comparación con los sistemas TCO de dominio de tiempo de la primera generación. Nota: El sistema octubre se interconecta con una sonda de fibra óptica, la exploración helicoidal en ~ 90 ° ángulo. Tiene un diámetro exterior de 2.7F (0,9 mm) y una longitud insertable de 135 cm. La sonda se conecta a la consola de octubre hasta un motor de accionamiento y el controlador óptico (muelle de montaje) con un rango de retroceso de 54 mm. Los conjuntos de datos de OCT adquiridas constan de 541 imágenes de cortes transversales (B-scan) wITH una resolución axial de 15 mm (Figura 1, 2). </li><li> Para garantizar mediciones de atenuación exactos y reproducibles, calibrar mediante la medición μ PTU para su aumento de las concentraciones con base en porcentaje en peso de una emulsión de grasa, (por ejemplo, Intralipid) como se describe anteriormente por Kodach et al. 14, 15. En breve: <ol><li> Diluir un lote estándar de 20% de emulsión de grasa con desmineralizada H 2 O para alcanzar concentraciones de 0,125, 0,250, 0,5, 1,0, 2,0, 4,0, 10, 15 y 20 (de stock) por ciento. <ol><li> Coloque la sonda octubre en 200 ml de mezcla de emulsión de grasa y adquirir una medición de octubre. </li><li> Referencia cruzada extraído μ octubre valores con valores conocidos en la literatura. </li></ol></li></ol></li></ol> 2. Time Out y Colocación del paciente <ol><li> Antes de iniciar el procedimiento, realice un &quot;tiempo fuera&quot; comprobar el nombre, fecha de nacimiento, procedimiento, prlado ocedural, uso de anticoagulantes, y las alergias. </li><li> Dependiendo de la localización del tumor, colocar al paciente en cualquier posición decúbito prono o lateral. Proporcionar al paciente con un apoyo adecuado y verificar si él / ella espera estar cómodo en esta posición durante un período de 20 a 40 min. </li><li> El uso de ultrasonido (US) 16, la localización del tumor y marcar el punto de entrada de la aguja en la piel con tinta permanente. NOTA: Cuando se utiliza la tomografía computarizada (TC), utilice una plantilla orientación aguja flexible para localizar la posición preferente de la aguja de acceso. </li></ol> 3. Desinfección y estéril Draping <ol><li> Ponte una gorra y la boca cubierta quirúrgica. </li><li> Limpie la piel alrededor del sitio de la punción utilizando una solución de clorhexidina / alcohol, teniendo cuidado de no quitar la marca de entrada de la aguja colocada previamente (paso 2.3). Desinfección de un área amplia evitará la necesidad de una limpieza adicional en caso de inesperado reposicionamiento aguja de acceso. </li><li> Con reGard del contenido estéril, abrir el conjunto de la punción percutánea que contiene: una jeringa de 10 ml, una aguja de aspiración romo, un G aguja de inyección 21, un bisturí, una aguja introductora co-axial 15 G, G una aguja trocar 18 y un 16 G pistola de biopsia de núcleo. </li><li> Lávese bien las manos, aplicar desinfectante de manos después. Ponte una bata quirúrgica y guantes estériles. </li><li> Cubra al paciente en paños estériles. </li><li> Aplique una cubierta estéril alrededor de la sonda de ultrasonido y fijar la guía de la aguja en su lugar. </li></ol> 4. Preparación octubre <ol><li> Inicie la consola de octubre e introduzca los datos del paciente en los campos marcados identificación del paciente, apellido, primer nombre y DOB (fecha de nacimiento), utilizando la interfaz de la consola. </li><li> Respecto del contenido estéril, descomprimir el paquete de octubre que contiene una sonda de octubre, una cubierta de muelle de montaje estéril y una jeringa luer-lock 5 ml. </li><li> Aplicar la cubierta estéril hasta el muelle de montaje de la consola octubre. Guiando el muelle de montaje no estéril requiere laayuda de un asistente. </li><li> Llene la jeringa de 5 ml con NaCl al 0,9% y adjuntarlo al puerto de descarga. Enjuague la sonda de octubre hasta que aparezca agua en la parte distal de la cubierta de la sonda. </li><li> Cargue la sonda de octubre en la base de montaje. Después de cargar la sonda girará y emitir luz roja de confirmar su correcto funcionamiento. Deja la sonda en su cubierta protectora durante el lavado y la carga para reducir al mínimo el riesgo de daños. </li><li> Retire la sonda de octubre de su cubierta. Coloque la sonda sobre una superficie dura y el uso de un bisturí para acortar la punta. Fijar la parte distal de la sonda durante el corte con el fin de minimizar la presión sobre la fibra óptica y el prisma. Cortar 5 mm distal del prisma, utilizando la (rojo) luz emitida para la orientación. </li></ol> 5. Punción <ol><li> Anestesiar la piel y las capas profundas utilizando lidocaína al 2% (20 mg / ml). Espere unos minutos que permitan la lidocaína para tener efecto. Pregunte al paciente si hay algún dolor. </li><li> Uso de la guía de la aguja, coloque la15 G co-axial la aguja introductora la verificación de la posición a través de imágenes. Si la colocación es satisfactoria, retire el obturador (núcleo aguja). </li><li> Coloque el G aguja trocar 18 a través de la aguja introductora, la perforación de la tumor. Una vez más verificar la posición de la aguja con formación de imágenes. Si la colocación es satisfactoria retirar el obturador. </li><li> Alimentar a la sonda de octubre hasta la aguja trocar hasta sentir resistencia. </li><li> Mientras que la fijación de la sonda de PTU, retraer la aguja trocar, dejando al descubierto la sonda OCT para el tejido tumoral. Manteniendo la punta de la aguja trocar dentro del tumor minimiza el retorcimiento de la sonda de PTU durante ciclos de respiración. Esto reduce el riesgo de daño de la sonda. </li><li> Octubre Scan: <ol><li> Realizar una exploración octubre, con la consola fijado en 541 B-scan por conjunto de datos. El sistema OCT utiliza aquí llevará a cabo una retirada automatizada en una longitud de 5,4 cm que no requieren ajustes de parámetros específicos. </li><li> Compruebe la exploración de calidad, artefactos y la aparición de tejido sólido (Figura 1A). Los artefactos más comúnmente aparecen como bandas circulares de pie entre el patrón normal octubre (Figura 1B). </li><li> Vuelva a colocar la sonda si persiste después de volver a analizar artefactos. </li></ol></li><li> Repita el paso 5.6 hasta un mínimo de 3 conjuntos de datos de OCT se adquieren. </li><li> Retire la sonda PTU y aguja trocar, dejando la aguja introductora en el lugar. </li><li> Armar el arma biopsia central y colocarlo a través de la aguja introductora, la verificación de la posición en la imagen. </li><li> Si la colocación es satisfactoria, disparar el arma biopsia. </li><li> Coloque el material de biopsia en el recipiente según el protocolo departamento de patología. Aquí, lugar biopsias en una placa de Petri con una incrustación de papel, suficientemente saturados con 0,9% de NaCl. </li><li> Compruebe la calidad de la biopsia central y repita el paso 5.9 y 5.10 hasta obtener material suficiente. </li></ol>

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	<li>Jemal, A., Siegel, R., Xu, J., Ward, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2010.">Cancer statistics, 2010.</a> CA Cancer J. Clin. 60, 277-300 (2010).</li><li>Mathew, A., Devesa, S. S., Fraumeni, J. F., Chow, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+increases+in+kidney+cancer+incidence,+1973-1992.">Global increases in kidney cancer incidence, 1973-1992.</a> Eur. J. Cancer Prev. 11, 171-178 (2002).</li><li>Volpe, 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=Contemporary+management+of+small+renal+masses.">Contemporary management of small renal masses.</a> Eur. 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The authors of this article have nothing to disclose.

En esta publicación nos informe sobre la viabilidad de la percutánea, la aguja basada, octubre del riñón. Este es un primer paso esencial en el desarrollo de octubre en una técnica aplicable clínicamente para la diferenciación del tumor, denominado como una &quot;biopsia óptica&quot;. Nuestros primeros 25 pacientes han demostrado percutánea octubre al ser un procedimiento sencillo y seguro. Una biopsia óptica tiene dos ventajas sobre biopsias convencionales. En primer lugar, la adquisición en tiempo real y an...

Las últimas décadas han mostrado un aumento constante en la incidencia de tumores renales 1,2. Hasta ahora, las decisiones de tratamiento en masa renal se han realizado principalmente en la base de MRI y CT características de imagen, la edad y la comorbilidad. Sin embargo, estos métodos de diagnóstico y los parámetros clínicos carecen de la sutileza para detectar realmente el potencial de malignidad de una masa renal. Una biopsia de núcleo o aspiración con aguja fina con el tejido suficiente para la evaluación patológica (de diagnóstico) proporciona la diferenciación tumoral objetiva tanto con la sensibilidad y especificidad en el rango de 95-100% 3. Por lo tanto la biopsia está ganando aceptación en la evaluación de las masas renales sospechosas 4,5. Sin embargo, las biopsias sin tejido suficiente para establecer un diagnóstico o con normalidad parénquima renal (no diagnóstico) se producen a un ritmo del 10-20% en general, e incluso hasta un 30% en pequeñas masas renales (&lt;4 cm, MER), lo que retrasa el proceso de diagnóstico debido a la necesidad frecuente para adicionalprocedimientos de biopsia de 3,5. La tomografía de coherencia óptica (OCT) es una novela técnica de imagen que tiene el potencial para superar los obstáculos mencionados en la diferenciación de la masa renal. Basado en la retrodispersión de la luz del infrarrojo cercano, OCT proporciona imágenes con una resolución axial 15 micras, con una penetración en el tejido eficaz de 2-3 mm (Figura 1, 2). La pérdida de intensidad de la señal por milímetro de penetración en el tejido, una resultante de los tejidos específicos de dispersión de la luz, se expresa como el coeficiente de atenuación (μ OCT: -1 mm) como se describe por Faber et al 6.. Características histológicas se pueden correlacionar con los valores μ octubre proporcionar un parámetro cuantitativo para la diferenciación de tejidos (Figura 3). Durante la carcinogénesis, las células malignas muestran un número aumentado, los núcleos más grandes y con forma irregular<html> un mayor índice de refracción y las mitocondrias más activos. Debido a esta sobreexpresión de componentes de la célula, un cambio en μ OCT es de esperar cuando la comparación de los tumores malignos de tumores benignos o tejido no afectado 7. Recientemente se ha estudiado la capacidad de la TCO superficial para diferenciar entre masas renales benignos y malignos 8,9. En 16 pacientes, intraoperatorias mediciones octubre de tejido tumoral se obtuvieron utilizando una sonda de octubre colocado externamente. El grupo de control compuesto por mediciones octubre de tejido no afectado en los mismos pacientes. El tejido normal mostró un coeficiente de atenuación mediana significativamente menor en comparación con el tejido maligno, lo que confirma el potencial de la OCT para la diferenciación del tumor. Este análisis cuantitativo se ha aplicado de una manera similar a la de grado otros tipos de tejido maligno, tales como carcinoma urotelial 10,11 y vulvar diferenciación neoplasia epitelial 12. ent &quot;&gt; Nuestro objetivo es desarrollar octubre en una biopsia óptica, que proporciona imágenes en tiempo real combinado con la diferenciación del tumor en el terreno. El objetivo del presente estudio es describir un percutánea, aguja basada, octubre enfoque en pacientes diagnosticados con un masa renal mejorador sólido. Esta descripción del método es, a nuestro entender, el primero en evaluar la posibilidad de aguja octubre con base de tumores renales.

<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.

Entre los primeros 25 tumores (23 pacientes), se realizaron un total de 24 procedimientos exitosos octubre. En un caso de un mal funcionamiento de la sonda llevó a la incapacidad para adquirir una exploración de PTU. Dos eventos adversos (AE) se produjo, que se describen en detalle en la sección de discusión. Características generales de los pacientes se encuentran en la Tabla 1. La consola de octubre ha software pre-instalado que proporciona en tiempo real las imágene...

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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.

En Vivo, percutánea, Aguja Based, tomografía de coherencia óptica de las masas renales

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 vistas.  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 tomografía de coherencia óptica de la circulación retiniana

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

Investigación

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.

Cerca de la tomografía de proyección óptica por infrarrojos para las Evaluaciones de distribución de la masa de células β en Investigación de la Diabetes

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.

Generación y cuantificación de 3 dimensiones de las lesiones arteriales en ratones mediante tomografía óptica de proyección

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.

En vivo de imágenes óptico de tumores del cerebro y la artritis Usando fluorescente SAPC-DOPS nanovesículas

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.

Evaluación de Preparación y respirometría de mitocondrias aisladas de tejido músculo esquelético Obtenidos percutánea Biopsia con aguja

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.

Evaluación transcutánea de la función renal en roedores 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.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

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.

Tomografía de coherencia óptica ultraalta resolución ratón para facilitar la inyección Intraocular en investigación de retina la terapia génica

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.

En Vivo Proyección de imagen de ratones de reportero Cx3cr1gfp/gfp con tomografía de coherencia óptica de dominio espectral y exploración oftalmoscopia de láser

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.

Evaluación de la función renal en modelos de ratón de la enfermedad Glomerular

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