Esta investigación fue financiada por la National Science Foundation Grant No. 0651981 y el Alexander y Margaret Stewart confianza, aunque el Centro de Cáncer de la Universidad Johns Hopkins. Los autores desean agradecer la contribución de la Dra. Rhoda Alani a la IRB y el estudio del paciente, así como la ayuda y el apoyo del Dr. Sewon Kang y su departamento durante el estudio del paciente.

1. Configuración <ol><li> A temperatura ambiente controlado examen equipado con una cámara infrarroja y un PC para la adquisición de imágenes de infrarrojos y almacenamiento, así como una tarjeta de adquisición de datos conectada a un ordenador se muestran en la Fig.1a. </li><li> La temperatura ambiente y temperatura de superficie de la piel son supervisados ​​por los termopares conectados a una tarjeta de adquisición de datos durante el estudio del paciente y los datos de medición se almacenan en el ordenador. </li></ol> 2. Adquisición de imágenes <ol><li> Dado que la lesión no se puede detectar en la imagen térmica, sin el efecto de enfriamiento, un marcador adhesivo cuadrado se utiliza para localizar la lesión pigmentada de interés y su entorno (fig. 1b). </li><li> Adquirimos una imagen de luz brillante de la lesión pigmentada y la ventana de adhesivo con una cámara digital (Canon PowerShot G11) (Fig. 1B). </li><li> A dermatoscopio conectado a una cámara digital (DermLite Foto System) se utiliza para capturar la imagen de la luz polarizada. </li><li> Adquirimos una imagen de estado de infrarrojos constante con un midwave Merlín (3-5 micras) se muestra en la cámara de infrarrojos Fig.1a, c. </li><li> Nosotros aplicamos una corriente de aire frío en el área de la piel del paciente que contiene la lesión, así como una región de 50 mm de diámetro alrededor de la duración de un minuto. </li><li> Después de un minuto, la eliminación de esta tensión de enfriamiento para permitir que la piel vuelva a calentar a temperatura ambiente dentro de 3-4 minutos (fase de recuperación térmica) (Fig. 1c-d). </li><li> Durante la fase de recuperación térmica, las imágenes infrarrojas de la lesión pigmentada son capturados cada 2 segundos (Fig. 1c-d). </li><li> Todas las imágenes de infrarrojos (además de la luz blanca y luz polarizada imágenes) tomadas durante el estudio se guardan y se almacenan utilizando el software Labview. </li></ol> 3. Procesamiento de imágenes <ol><li> Las imágenes IR se analizaron mediante un código de Matlab dedicado con el fin de obtener información precisa distribución de temperatura transitoria en la superficie de la piel. Para ello, se introducen diversas medidas de calibración y un sistema de análisis de imagen multimodal. </li><li> Comenzamos con la aplicación de un algoritmo de detección sin precedentes para la imagen de luz brillante para la localización de las esquinas del marcador adhesivo. A continuación, hemos identificado los puntos correspondientes en la imagen de referencia IR. </li><li> Con el fin de compensar involuntarios del cuerpo / extremidades movimiento del paciente, se utilizan estos puntos como puntos de referencia en un modelo de movimiento de segundo grado para la alineación de la secuencia de imágenes de infrarrojos durante la fase de recuperación. </li><li> Usamos el andador al azar, un algoritmo de segmentación de imágenes interactivas donde el usuario puede orientar espacialmente la segmentación mediante la colocación de puntos de la semilla, para crear una imagen de la máscara de delinear la lesión. </li><li> Una vez que se determina la forma de la lesión, identificamos la zona correspondiente de cada una de las registradas las imágenes de infrarrojos. </li><li> Se selecciona al azar los puntos dentro de la lesión y lejos de la lesión en representación de la lesión y el tejido sano, respectivamente. </li><li> Comparamos la respuesta transitoria térmica de la piel sana y la respuesta de la lesión. </li><li> Se prepara una tabla que muestra todos los datos: digital, la dermatoscopia, con código de color las imágenes infrarrojas de la lesión y sus alrededores registraron en las condiciones ambientales y 2 segundos después de la excitación de refrigeración, y la respuesta transitoria termal de la lesión y el tejido sano. </li></ol> 4. Los resultados representativos: <img src="/files/ftp_upload/2679/2679fig1.jpg" alt="Figura 1" /> Figura 1. a) El sistema de imágenes por infrarrojos HRIS en la sala de ensayo clínico, b) fotografía de la superficie corporal más grande, con un grupo de lesiones pigmentadas y el marco de plantilla aplicada para imágenes, c) la referencia de la imagen infrarroja de la región a temperatura ambiente, d) la misma zona después del enfriamiento y e) la sección ampliada de la lesión de melanoma y su entorno <img src="/files/ftp_upload/2679/2679fig2.jpg" alt="Figura 2" /> Figura 2. Sala de examen con nuestro sistema de imágenes térmicas. <img src="/files/ftp_upload/2679/2679fig3.jpg" alt="Figura 3" /> Figura 3. El enfriamiento de la lesión y el tejido de la piel circundante soplando una corriente de aire frío de un tubo de vórtice.

<ol>
	<li>Elder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+progression,+early+diagnosis+and+prognosis+of+melanoma.">Tumor progression, early diagnosis and prognosis of melanoma.</a> Acta Oncol. 38, 535-547 (1999).</li><li>Wartman, D., Weinstock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+overemphasizing+sun+avoidance+in+protection+from+melanoma.">Are we overemphasizing sun avoidance in protection from melanoma.</a> Cancer Epidemiol Biomarkers Prev. 17, 469-470 (2008).</li><li>Pirtini Cetingul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using high resolution infrared imaging to detect melanoma and dysplastic nevi [dissertation]. , (2010).</li><li>Jones, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reappraisal+of+the+use+of+infrared+thermal+image+analysis+in+medicine.">A reappraisal of the use of infrared thermal image analysis in medicine.</a> IEEE Trans. Med. Imaging. 17, 1019-1027 (1998).</li><li>Anbar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+thermal+imaging+today-shifting+from+phenomenological+thermography+to+pathophysiologically+based+thermal+imaging.">Clinical thermal imaging today-shifting from phenomenological thermography to pathophysiologically based thermal imaging.</a> IEEE Eng. Med. Biol. Mag. 17, 25-33 (1998).</li><li>Anbar, M., Gratt, B. M., Hong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermology+and+facial+telethermography.+Part+I:+history+and+technical+review.">Thermology and facial telethermography. Part I: history and technical review.</a> Dentomaxillofacial Radiology. 27, 61-67 (1998).</li><li>Jones, B. F., Plassmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+infrared+thermal+imaging+of+human+skin.">Digital infrared thermal imaging of human skin.</a> IEEE Eng. Med. Bio. 21, 41-48 (2002).</li><li>Qi, H., Diakides, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Infrared imaging in Medicine. , (2007).</li><li>Pirtini Cetingul, M., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+skin+lesions+from+the+transient+thermal+response+using+infrared+imaging+technique.">Identification of skin lesions from the transient thermal response using infrared imaging technique.</a> IEEE 5th Int. Symp. on Biomedical Imaging: From Nano to Macro 1-4. , 1219-1222 (2008).</li><li>Cetingul, P. i. r. t. i. n. i., M, ., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+thermal+signature+of+a+melanoma+lesion.">Quantification of the thermal signature of a melanoma lesion.</a> Int. Journal of Thermal Science. 50, 421-431 (2011).</li><li>Pirtini Cetingul, M., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heat+transfer+model+of+skin+tissue+for+the+detection+of+lesions:+sensitivity+analysis.">A heat transfer model of skin tissue for the detection of lesions: sensitivity analysis.</a> Physics in Medicine and Biology. 55, 5933-5951 (2010).</li><li>Pirtini Cetingul, M., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+skin+lesions+using+transient+thermal+imaging.">Quantitative evaluation of skin lesions using transient thermal imaging.</a> Proc. Int. Heat Transfer Conf. , (2010).</li></ol>

Los resultados sugieren que, al aplicar la tensión de enfriamiento que mejora las diferencias de temperatura entre la lesión y el tejido sano circundante. Además, debido a pequeños movimientos del paciente durante la termografía, tuvimos que aplicar el seguimiento del movimiento de la superposición correctamente las imágenes para medir las diferencias de temperatura entre el estado de referencia y la distribución de la temperatura durante la recuperación térmica. Sin el control de movimiento que no han sido capaces de detectar y medir la diferencia de temperatura entre la lesión maligna y el tejido sano. Estos resultados y la necesidad de movimiento de un seguimiento preciso explicar las dificultades que enfrentan los investigadores en el pasado al tratar de diagnosticar el melanoma utilizando imágenes de IR sobre la base de información de estado estable y solo demuestran claramente las ventajas de la termografía dinámica. Cabe señalar que la resolución espacial de la cámara de infrarrojos (número de píxeles en la matriz de plano focal IR) es fundamental cuando las lesiones pequeñas exigentes. Tanto la resolución espacial y la sensibilidad a la temperatura de las cámaras infrarrojas temprano fue limitado, lo que también da cuenta de las dificultades en la detección de melanoma en estadio temprano en el pasado. Las principales diferencias entre nuestro enfoque y antes de los intentos de imagen térmica - que fueron un éxito moderado - son las secuencias de calibración y medidas de procesamiento de imágenes que nos permiten medir con precisión las diferencias de temperatura en este sistema, además del proceso de imágenes dinámicas que se basa en la refrigeración activa.

<table border="1"><tbody><tr><td> Nombre del equipo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Merlin MWIR cámara </td><td> FLIR </td><td> - </td></tr><tr><td> Canon PowerShot G11 </td><td> Canon </td><td> - </td></tr><tr><td> DermLite Foto Sistema </td><td> DermLite </td><td> - </td></tr><tr><td> Vortex tubo </td><td> Exair </td><td> - </td></tr><tr><td> Los tanques de aire </td><td> Airgas </td><td> - </td></tr></tbody></table>

quantitative visualization and detection of skin cancer using dynamic thermal imaging

En 2010, aproximadamente 68.720 melanomas se diagnostican en los EE.UU., con alrededor de 8.650 con resultado de muerte 1. Hasta la fecha, el único tratamiento eficaz para el melanoma sigue siendo la resección quirúrgica, por lo tanto, la clave para la supervivencia extendida es la detección precoz 2,3. Teniendo en cuenta el gran número de pacientes diagnosticados cada año y las limitaciones en el acceso a la atención especializada con rapidez, el desarrollo de objetivos en vivo los instrumentos de diagnóstico para ayudar en el diagnóstico es esencial. Nuevas técnicas para detectar el cáncer de piel, sobre todo herramientas no invasivas de diagnóstico, se están estudiando en muchos laboratorios. Junto con los métodos de las técnicas quirúrgicas, como la fotografía digital, la dermatoscopia, sistemas multiespectrales de imágenes (MelaFind), sistemas basados ​​en láser (la microscopía confocal de barrido láser, láser doppler imágenes de perfusión, tomografía de coherencia óptica), ultrasonido, resonancia magnética, se están probando . Cada técnica ofrece ventajas e inconvenientes, muchos de los cuales representan un compromiso entre la eficacia y la precisión frente a la facilidad de uso y el coste. Los detalles sobre estas técnicas y las comparaciones están disponibles en la literatura 4. Infrarrojos (IR) de imágenes ha demostrado ser un método útil para diagnosticar los síntomas de ciertas enfermedades mediante la medición de la temperatura local de la piel. Hay un gran cuerpo de evidencia que muestra que la enfermedad o la desviación de funcionamiento normal se acompaña de cambios de la temperatura del cuerpo, que a su vez afectan a la temperatura de la piel 5,6. Datos precisos sobre la temperatura del cuerpo humano y la piel pueden proporcionar una gran cantidad de información sobre los procesos responsables de la generación de calor y la termorregulación, en particular la desviación de las condiciones normales, a menudo causada por la enfermedad. Sin embargo, la imagen IR no ha sido ampliamente reconocida en la medicina debido a la utilización prematura de la tecnología de 7,8 hace varias décadas, cuando la exactitud de medición de temperatura y la resolución espacial fueron las herramientas de imagen inadecuada y sofisticado procesamiento no estaban disponibles. Esta situación cambió drásticamente a finales de los 1990s-2000s. Los avances en la instrumentación de infrarrojos, la aplicación de la tecnología digital y algoritmos de procesamiento dinámico de imágenes de infrarrojos, que permite a los científicos para analizar no sólo el espacio, sino también el comportamiento temporal térmica de la piel 9, los avances permitidos en el campo. En nuestra investigación, analizamos la viabilidad de la imagen IR, junto con estudios teóricos y experimentales, como un costo efectivo, no invasivo, en la técnica de medición óptica in vivo para la detección del tumor, con énfasis en el cribado y la detección precoz del melanoma 10-13 . En este estudio, se muestran los datos obtenidos en un estudio de pacientes en los que los pacientes que tienen una lesión pigmentada con una indicación clínica de la biopsia son seleccionados para la imagen. Se comparó la diferencia en las respuestas térmicas entre el tejido sano y malignos y comparamos nuestros datos con los resultados de la biopsia. Llegamos a la conclusión de que la actividad metabólica de la lesión de melanoma pueden ser detectados por imágenes por infrarrojos dinámica.

Watch this Scientific Journal Video about Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging at JoVE.com

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

Hemos demostrado que las lesiones pigmentadas malignas con la actividad metabólica de generar cantidades cuantificables de calor y la medición de la respuesta transitoria térmica de la piel a una excitación de enfriamiento permite la identificación cuantitativa de melanoma y otros cánceres de piel (vs. no-proliferación nevus) a una edad temprana etapa de la enfermedad.

Visualización y detección cuantitativa de cáncer de piel utilizando imágenes térmica dinámica

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

quantitative-visualization-detection-skin-cancer-using-dynamic

Research

JoVE Journal

Medicine

16.6K vistas.  The Johns Hopkins University. Hemos demostrado que las lesiones pigmentadas malignas con la actividad metabólica de generar cantidades cuantificables de calor y la medición de la respuesta transitoria térmica de la piel a una excitación de enfriamiento permite la identificación cuantitativa de melanoma y otros cánceres de piel (vs. no-proliferación nevus) a una edad temprana etapa de la enfermedad.

Video: Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

La medición precisa y simple de la citoquina pro-inflamatoria IL-1β mediante un análisis de sangre entera estimulación

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Investigación

Medicina

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Análisis cuantitativo de la metástasis del cáncer utilizando un modelo de embrión aviar

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

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. ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

Análisis Cuantitativo multiespectrales después de un trasplante de tejido fluorescente para la visualización de los Orígenes celulares, tipos y Interacciones

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

Líquido cefalorraquídeo MicroRNA perfiles utilizando cuantitativa en tiempo real PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to clearly describe the acromion marker cluster (AMC) method of determining scapular kinematics when using a passive marker motion capture system, with consideration for the sources of error which could affect the validity and reliability of measurements. The AMC method involves placing a cluster of markers over the posterior acromion, and through calibration of anatomical landmarks with respect to the marker cluster it is possible to obtain valid measurements of scapular kinematics. The reliability of the method was examined between two days in a group of 15 healthy individuals (aged 19-38 years, eight males) as they performed arm elevation, to 120&#176;, and lowering in the frontal, scapular and sagittal planes. Results showed that between-day reliability was good for upward scapular rotation (Coefficient of Multiple Correlation; CMC = 0.92) and posterior tilt (CMC = 0.70) but fair for internal rotation (CMC = 0.53) during the arm elevation phase. The waveform error was lower for upward rotation (2.7&#176; to 4.4&#176;) and posterior tilt (1.3&#176; to 2.8&#176;), compared to internal rotation (5.4&#176; to 7.3&#176;). The reliability during the lowering phase was comparable to results observed during the elevation phase. If the protocol outlined in this study is adhered to, the AMC provides a reliable measurement of upward rotation and posterior tilt during the elevation and lowering phases of arm movement.

This report presents details of how to adopt the acromion marker cluster method of obtaining scapular kinematics when using a passive marker motion capture device. As has been described in the literature, this method provides a robust, non-invasive, three-dimensional, dynamic and valid measurement of scapular kinematics, minimizing skin movement artifact.

La medición de la Dinámica Escapulario Cinemática El uso de un Cluster Acromion marcador para Minimizar Artefacto Movimiento Piel

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic Contrast mejorada de imagen de resonancia magnética de un modelo ortotópico de cáncer pancreático del ratón

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Espectroscopia óptica difusa para la evaluación cuantitativa de la radiación ionizante aguda La toxicidad cutánea inducida utilizando un modelo de ratón

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualización Cuantitativa del Infiltrado de Leucocitos en un Modelo Murino de Miocarditis Fulminante por Microscopía de Hoja Ligera

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Cuantitativo [18F]-Naf-PET-MRI Análisis para la evaluación de la rotación ósea dinámica en un paciente con dolor de espalda baja facetogénico

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...