Estamos en deuda con la señora Ina Wesselman para la formación y la planificación del personal de quirófano.

1. Intraoperatoria cámara multiespectral de fluorescencia <ol><li> Un sistema de cámara de medida se ha desarrollado en el desarrollado en el Instituto de Medicina Biológica y de imagen (IBMI, de la Universidad Técnica / Helmholtz Zentrum, Munich, Alemania), en estrecha en colaboración con SurgOptix (SurgOptix Inc., Redwood Shores, California, EE.UU.). La configuración del sistema de cámara multiespectral intraoperatoria de fluorescencia se muestra en la Figura 2. </li><li> La luz pasa a través de un sistema de óptica y se divide en luz visible, luz en la banda de longitudes de onda de emisión (fluorescencia) y la luz en la banda de longitudes de onda de excitación. Estos paquetes son detectados por cámaras CCD (CCD) (Figura 2). Multi-espectral de señales de todas las cámaras son procesados ​​con el fin de corregir los artefactos y el rendimiento real cuantitativa fluorocromo bio-distribución. Color y las señales de fluorescencia se puede visualizar como imágenes separadas en monitores externos o superpuestas en una sola imagen. </li><li> Los filtros de excitación y emisión de la cámara se establecen en el documento de medición para recoger a 750 nm y 800 nm ± 20 respectivamente. </li></ol> 2. Óptica de contraste de la preparación del agente <ol><li> El agente de contraste fluorescente verde de indocianina (ICG, Pulsion AG, Munich, Alemania) se prepara en condiciones estériles. Una concentración de 0,5 mg / ml se utiliza. Es importante el uso de agua destilada estéril y no cloruro de sodio (NaCl), ya que ésta hará que el ICG para agregar. </li><li> Después de la preparación, la solución debe ser almacenado en un lugar oscuro y fresco para evitar el deterioro rápido de la intensidad de la fluorescencia por el blanqueo. Nota: a partir de ahora mantener el ICG protegido de la luz. </li></ol> 3. Las imágenes intraoperatorias - Configuración e inyección de ICG <ol><li> La cámara se coloca y se inició en la sala de operaciones antes de la cirugía para minimizar la interferencia con el procedimiento quirúrgico estándar, y conectados a las pantallas de alta definición (Figura 2B). </li><li> La cámara está cubierto en la norma paños estériles (Carl Zeiss Vision BV, Sliedrecht, Países Bajos, REF OPMI Coloque 306.071) por el investigador en estéril o la ropa. </li><li> Nota: se tiene cuidado de utilizar sólo los agentes de desinfección claro, como color de los agentes desinfectantes son a menudo autofluorescentes y podría interferir con el procedimiento de imagen. Por otra parte, paños estériles y los marcadores quirúrgica puede ser autofluorescentes. Se recomienda probar todos los materiales utilizados en el campo de la O y la operación de autofluorescencia antes de la cirugía. </li><li> El agente de contraste fluorescente verde de indocianina (ICG) se prepara en condiciones estériles. Una concentración de 0,5 mg / ml se utiliza. Es importante el uso de agua destilada estéril y no NaCl, ya que ésta hará que el ICG para agregar. Después de la preparación, la solución debe ser almacenado en un lugar oscuro y fresco para evitar el deterioro rápido de la intensidad de la fluorescencia. Nota: es recomendable cambiar los guantes después de la preparación de ICG, ya que incluso una pequeña cantidad de derrame de ICG puede influir en la calidad de la imagen. </li><li> El procedimiento de imágenes comienza después de abrir el abdomen. Cuando el área de interés está expuesta, la cámara se maneja en el campo de operación. El zoom y el enfoque se ajusta en condiciones estériles. Luces en la sala de operaciones se apagan para una mejor detección de la señal de fluorescencia. </li><li> 1,0 ml de 0,5 mg / ml de ICG se mezcla con 1,0 ml de la norma de colorante azul (azul patente / azul patente, Guerbet, Francia) en una jeringa. El cirujano inyecta el medio de contraste en los cuatro cuadrantes alrededor del tumor primario, la prevención de derrames del agente. Nota: en caso de derrame de ICG en los guantes quirúrgicos, es necesario cambiar los guantes. Las imágenes fijas o videos en tiempo real se obtienen del flujo de la linfa y la aparición de los ganglios linfáticos centinela fluorescente (Figura 3). Todas las imágenes y los vídeos se guardan directamente en el ordenador. </li><li> Aparte de la fluorescencia, la SLN se detecta también de acuerdo con el protocolo estándar, ya sea utilizando una sonda de rayos gamma o por inspección visual de la coloración azul, o ambas cosas. El trazador radioactivo se administra generalmente un día antes de la cirugía, después de que un lymphoscintigram se realiza para la detección de un ganglio centinela radiactivos. Esto es parte del procedimiento estándar y no tiene lugar en el protocolo de imágenes de fluorescencia. </li><li> En tiempo real de la escisión del ganglio centinela se guía tanto por la decoloración de fluorescencia y el azul de los ganglios linfáticos. La función en tiempo real de la cámara ayuda a detectar la fluorescencia de cualquier residuo o localizaciones inesperadas de los nodos de fluorescentes. </li><li> Después de la extirpación del ganglio centinela, ex vivo las imágenes se adquieren de todos los ganglios linfáticos extirpados de la presencia de una señal fluorescente. El tiempo de exposición puede ser prolongada para una mayor resolución en la captura de imágenes fijas (Figura 4). </li><li> El examen histopatológico de los ganglios linfáticos revela la presencia o ausencia de células tumorales en el ganglio linfático centinela. </li></ol> 4. RResultados epresentante Ganglios linfáticos fluorescentes se pueden detectar con una elevada relación señal-a-fondo de relación. Además, el flujo de ICG a través de los vasos linfáticos pueden ser monitoreados en tiempo real que permite la cartografía de los ganglios linfáticos. Los resultados pueden ser influenciados por aumentar la profundidad del nodo, que posiblemente requieran procedimientos diferentes de iluminación para lograr la detección. La aplicación futura de tumor objetivo específico agentes fluorescentes pueden proporcionar una detección intraoperatoria de un ganglio centinela positivo con células de cáncer con esta tecnología. <img src="/files/ftp_upload/2225/2225fig1.jpg" alt="Figura 1" /> Figura 1. El ganglio centinela (GC) la teoría. SLN es el primer ganglio de drenaje linfático (s) del tumor. <img src="/files/ftp_upload/2225/2225fig2.jpg" alt="Figura 2" /> Figura 2. Esquema de imagen del sistema de cámara de fluorescencia multiespectral (A). La configuración básica del sistema de cámaras en el quirófano (B). <img src="/files/ftp_upload/2225/2225fig4.jpg" alt="Figura 3" /> Figura 3. Imágenes multiespectrales de fluorescencia de un ganglio linfático en el cáncer de vulva. La imagen a color de un ganglio linfático en vivo (A). Imágenes de fluorescencia de los ganglios linfáticos mismo, en vivo (B). Pseudocolor de imágenes de fluorescencia superpuesta a la imagen en color (C). <img src="/files/ftp_upload/2225/2225fig5.jpg" alt="Figura 4" /> Figura 4. Imágenes multiespectrales de fluorescencia de un ex vivo linfáticos ganglios en el cáncer de cuello de útero. La imagen a color de un ganglio linfático ex vivo (A). Imágenes de fluorescencia de los ganglios linfáticos mismo, ex vivo (B). Pseudocolor de imágenes de fluorescencia superpuesta a la imagen en color (C).

<ol>
	<li>Tinga, D. J., Bouma, J., Aalders, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patients+with+squamous+cell+versus+adeno(squamous)+carcinoma+of+the+cervix,+what+factors+determine+the+prognosis.">Patients with squamous cell versus adeno(squamous) carcinoma of the cervix, what factors determine the prognosis.</a> Int J Gynecol Cancer. 2 (2), 83-91 (1992).</li><li>McMahon, C. J., Rofsky, N. M., Pedrosa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+metastases+from+pelvic+tumors:+anatomic+classification,+characterization,+and+staging.">Lymphatic metastases from pelvic tumors: anatomic classification, characterization, and staging.</a> Radiology. 254 (1), 31-46 (2010).</li><li>Zivanovic, O., Khoury-Collado, F., Abu-Rustum, N. R., Gemignani, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sentinel+lymph+node+biopsy+in+the+management+of+vulvar+carcinoma,+cervical+cancer,+and+endometrial+cancer.">Sentinel lymph node biopsy in the management of vulvar carcinoma, cervical cancer, and endometrial cancer.</a> Oncologist. 14 (7), 695-705 (2009).</li><li>Fuller, J., Guderian, D., Kohler, C., Schneider, A., Wendt, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+edema+of+the+lower+extremities+after+lymphadenectomy+and+radiotherapy+for+cervical+cancer.">Lymph edema of the lower extremities after lymphadenectomy and radiotherapy for cervical cancer.</a> Strahlenther Onkol. 184 (4), 206-211 (2008).</li><li>Ayhan, A., Celik, H., Dursun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+mapping+and+sentinel+node+biopsy+in+gynecological+cancers:+a+critical+review+of+the+literature.">Lymphatic mapping and sentinel node biopsy in gynecological cancers: a critical review of the literature.</a> World J Surg Oncol. 6, 53-53 (2008).</li><li>Tanis, P. J., Nieweg, O. E., Valdes Olmos, R. A., Rutgers, E. J., Kroon, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+sentinel+node+and+validation+of+the+technique.">History of sentinel node and validation of the technique.</a> Breast Cancer Res. 3 (2), 109-112 (2001).</li><li>Ghobashy, A. E. E. l., Saidi, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sentinel+lymph+node+sampling+in+gynaecological+cancers:+techniques+and+clinical+applications.">Sentinel lymph node sampling in gynaecological cancers: techniques and clinical applications.</a> Eur J Surg Oncol. 35 (7), 675-685 (2009).</li><li>Alford, R., Simpson, H. M., Duberman, J., Hill, G. C., Ogawa, M., Regino, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+organic+fluorophores+used+in+molecular+imaging:+literature+review.">Toxicity of organic fluorophores used in molecular imaging: literature review.</a> Mol Imaging. 8 (6), 341-354 (2009).</li><li>Themelis, G., Yoo, J. S., Soh, K. S., Schulz, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+intraoperative+fluorescence+imaging+system+using+light-absorption+correction.">Real-time intraoperative fluorescence imaging system using light-absorption correction.</a> J Biomed Opt. 14 (6), 064012-064012 (2009).</li></ol>

VN y GMvD es miembro del consejo científico de SurgOptix Inc. Antes del procedimiento, el protocolo de investigación aprobado por la Junta de Revisión de locales de investigación (IRB) y el consentimiento informado del paciente se adquiere.

Este video muestra la aplicación de la fluorescencia de imágenes multiespectrales intraoperatoria tecnología para la detección intraoperatoria del ganglio centinela (GC) en oncología ginecológica. La metodología tiene ciertas ventajas sobre el procedimiento de ganglio centinela convencionales. La inyección se lleva a cabo durante el procedimiento quirúrgico en sí mismo con el paciente anestesiado en vez de un día antes de la cirugía, que es más fácil para el paciente, especialmente en los cánceres ginecológicos. Además, las imágenes intraoperatorias proporciona al cirujano feedback visual directa del flujo de la linfa y su patrón de drenaje de la GC, y no indirectamente a través de un contador Geiger-cajero. En el cáncer de cuello de útero, donde se encuentran los ganglios linfáticos en la pelvis, esto puede ayudar a mejorar la detección. Sin embargo, la mayor importancia radica en el enfoque paso hacia imágenes específicas. Dirigido agentes de contraste fluorescente puede ser fusionado con el tumor de anticuerpos específicos o sustratos dirigidas específicamente a las células tumorales. De esta manera, imagen de fluorescencia intraoperatoria tiene un gran potencial para alterar radicalmente la práctica actual de la cirugía oncológica.

A correction was made to <a href="http://www.jove.com/index/details.stp?id=2225">Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons</a>. There was an error with an author's name. The author name was corrected to include a first initial as follows:
K. Tim Buddingh
instead of:
Tim Buddingh

A correction to an authors name has been made for the article Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology.

Erratum: Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

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<td>Indocyanine Green (ICG)</td>
<td>&nbsp;</td>
<td>Pulsion Medical Systems AG, Munich, Germany</td>
<td>D-81829</td>
<td>A solution of ICG in distilled water is used and not in NaCl 0.9%.</td>
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<td>Patent blue / Bleu patent&eacute;</td>
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<td>Guerbet, Paris, France</td>
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<td>Sterile water for injection</td>
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<td>B. Braun</td>
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<td>&nbsp;</td>
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<td>Multispectral fluorescence camera system </td>
<td>&nbsp;</td>
<td>Institute for Biological and Medical Imaging (IBMI), Technical University, Munich, Germany and SurgOptix Inc, USA</td>
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multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology

El pronóstico en prácticamente todos los tumores sólidos depende de la presencia o ausencia de metástasis en los ganglios linfáticos. El tratamiento quirúrgico 1-3 más a menudo se combina la escisión radical del tumor con una linfadenectomía completa en el área de drenaje del tumor. Sin embargo, la extirpación de los ganglios linfáticos se asocia con aumento de la morbilidad debido a una avería infección de la herida, y el linfedema. 4,5 Como alternativa, el procedimiento de nodo linfático centinela (GC) se desarrolló hace varias décadas para la detección del primer ganglio de drenaje linfático del tumor 6. En el caso de la difusión de lymphogenic, el ganglio centinela es el primer ganglio linfático que se ve afectado (Figura 1). Por lo tanto, si el ganglio centinela no contiene metástasis, los ganglios linfáticos abajo también estará libre de metástasis del tumor y no tiene que ser eliminado. El procedimiento de ganglio centinela es parte del tratamiento para muchos tipos de tumores, como el cáncer de mama y melanoma, sino también para el cáncer de la vulva y el cuello uterino. 7 La metodología actual para la detección de ganglio centinela es la inyección peritumoral de radiocoloide un día antes de la cirugía, y un tinte de color durante la operación. Las desventajas de este procedimiento en cáncer cervical y vulvar son múltiples inyecciones en el área genital, lo que lleva a mayor angustia psicológica para el paciente, y el uso de materiales radiactivos coloide. Imágenes de fluorescencia multiespectral es una modalidad de imagen emergentes que se pueden aplicar durante la operación sin necesidad de inyección de radiocoloide. De imagen de fluorescencia intraoperatoria, dos componentes son necesarios: un agente fluorescente y un sistema cuantitativo óptica para las imágenes intraoperatorias. Como un fluoróforo que hemos utilizado con verde de indocianina (ICG). ICG ha sido utilizado durante décadas para evaluar la función cardíaca, la perfusión cerebral y la perfusión del hígado. 8 Es una droga inerte con un seguro fármaco-biológica perfil. Cuando se excita a 750 nm, que emite luz en el espectro del infrarrojo cercano a 800 nm. A medida multiespectrales sistema de fluorescencia de la cámara de imágenes se utilizó. 9. El objetivo de este artículo de video es para demostrar la detección del ganglio centinela intraoperatorio con fluorescencia de imagen en pacientes con cáncer cervical y vulvar. Fluorescencia de imagen se utiliza en conjunción con el procedimiento habitual, que consiste en radiocoloide y un colorante azul. En el futuro, imagen de fluorescencia intraoperatoria podría reemplazar el método actual y también es fácilmente transferible a otras indicaciones como el cáncer de mama y melanoma.

Watch this Scientific Journal Video about Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology at JoVE.com

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

Imagen de fluorescencia es una técnica prometedora para la innovadora cirugía guiada por imagen en cirugía oncológica. En este video se describe el procedimiento técnico para la detección del ganglio centinela utilizando imágenes de fluorescencia mostró como en ginecología oncologicy. Un sistema multiespectral cámara de fluorescencia, junto con el agente fluorescente verde de indocianina, se aplica.

Multiespectrales en tiempo real imágenes de fluorescencia para la detección intraoperatoria del ganglio centinela en Oncología Ginecológica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

multispectral-real-time-fluorescence-imaging-for-intraoperative

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23.0K vistas.  University Medical Center Groningen. Imagen de fluorescencia es una técnica prometedora para la innovadora cirugía guiada por imagen en cirugía oncológica. En este video se describe el procedimiento técnico para la detección del ganglio centinela utilizando imágenes de fluorescencia mostró como en ginecología oncologicy. Un sistema multiespectral cámara de fluorescencia, junto con el agente fluorescente verde de indocianina, se aplica.

Video: Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluación de la absorción de nanopartículas en los tumores en tiempo real utilizando intravital imagen

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Investigación

Medicina

Stereotactic Radiosurgery for Gynecologic Cancer

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, intricate treatment planning, on-board imaging, and reduced number of ablative radiation doses to cancer targets usually refractory to chemotherapy and conventional radiation. Steep SBRT radiation dose drop-off permits narrow 'pencil beam' treatment fields to be used for ablative radiation treatment condensed into 1 to 3 treatments.
Treating physicians must appreciate that SBRT comes at a bigger danger of normal tissue injury and chance of geographic tumor miss. Both must be tackled by immobilization of cancer targets and by high-precision treatment delivery. Cancer target immobilization has been achieved through use of indexed customized Styrofoam casts, evacuated bean bags, or body-fix molds with patient-independent abdominal compression.1-3 Intrafraction motion of cancer targets due to breathing now can be reduced by patient-responsive breath hold techniques,4 patient mouthpiece active breathing coordination,5 respiration-correlated computed tomography,6 or image-guided tracking of fiducials implanted within and around a moving tumor.7-9 The Cyberknife system (Accuray [Sunnyvale, CA]) utilizes a radiation linear accelerator mounted on a industrial robotic arm that accurately follows patient respiratory motion by a camera-tracked set of light-emitting diodes (LED) impregnated on a vest fitted to a patient.10 Substantial reductions in radiation therapy margins can be achieved by motion tracking, ultimately rendering a smaller planning target volumes that are irradiated with submillimeter accuracy.11-13
Cancer targets treated by SBRT are irradiated by converging, tightly collimated beams. Resultant radiation dose to cancer target volume histograms have a more pronounced radiation "shoulder" indicating high percentage target coverage and a small high-dose radiation "tail." Thus, increased target conformality comes at the expense of decreased dose uniformity in the SBRT cancer target. This may have implications for both subsequent tumor control in the SBRT target and normal tissue tolerance of organs at-risk. Due to the sharp dose falloff in SBRT, the possibility of occult disease escaping ablative radiation dose occurs when cancer targets are not fully recognized and inadequate SBRT dose margins are applied. Clinical target volume (CTV) expansion by 0.5 cm, resulting in a larger planning target volume (PTV), is associated with increased target control without undue normal tissue injury.7,8 Further reduction in the probability of geographic miss may be achieved by incorporation of 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET).8 Use of 18F-FDG PET/CT in SBRT treatment planning is only the beginning of attempts to discover new imaging target molecular signatures for gynecologic cancers.

Stereotactic body radiotherapy (SBRT) involves image-guided, ablative radiation delivered to cancer targets refractory to chemotherapy or to conventional radiation treatment. The robotic-armed Cyberknife SBRT system, using sophisticated target localization, delivers hypofractionated radiation doses capable of sterilizing cancer targets. This article will consider new therapeutic roles of SBRT for gynecological cancers.

La radiocirugía estereotáctica para el cáncer ginecológico

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Detección de microARN en tumores de próstata por el cuantitativo PCR en tiempo real (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Intraoperatoria detección de endometriosis sutil: un nuevo paradigma para la detección y tratamiento del dolor pélvico asociado con la pérdida de la integridad Peritoneal

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Cerenkov Luminiscencia Imaging (CLI) para la Vigilancia del Cáncer Terapia

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Trasplante en la cámara anterior del ojo para Longitudinal, no invasiva<em&gt; En vivo</em&gt; Imagen con una sola célula de resolución en tiempo real

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Inducida por la quimioterapia Vascular Toxicidad - En tiempo real<em&gt; En vivo</em&gt; Imagen de Deterioro del buque

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modelado de los primeros pasos de cáncer de ovario Difusión en una cultura organotípicos del Peritoneal Cavity Humano

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Prolongado lapso de tiempo intravital de imágenes de tiempo real multicelular Dinámica en el microambiente tumoral

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.