Esta pesquisa foi financiada pela National Science Foundation Grant No. 0651981 e Alexander Stewart e Margaret confiança que o Centro de Câncer da Universidade Johns Hopkins. Os autores gostariam de agradecer as contribuições do Dr. Rhoda Alani ao IRB e ao estudo do paciente, bem como a ajuda eo apoio do Dr. Sewon Kang e seu departamento durante o estudo paciente.

1. Instalação <ol><li> A temperatura ambiente controlada exame equipado com uma câmera infravermelha e um computador para aquisição de imagem de infravermelhos e armazenamento, bem como um cartão de aquisição de dados conectado a um computador são mostrados na Fig.1a. </li><li> A temperatura ambiente e temperatura da superfície da pele são monitoradas por termopares ligados a uma placa de aquisição de dados durante o estudo paciente e dados de medição são armazenados no computador. </li></ol> 2. Aquisição de imagem <ol><li> Uma vez que a lesão não pode ser detectado na imagem térmica, sem o efeito de resfriamento, um marcador adesivo quadrado é usado para localizar a lesão pigmentada de interesse e seu entorno (Figura 1b). </li><li> Nós adquirimos uma imagem de luz brilhante da lesão pigmentada ea janela adesivo com uma câmera digital (Canon PowerShot G11) (Figura 1b). </li><li> A dermatoscópio ligado a uma câmera digital (Foto DermLite System) é usado para capturar a imagem de luz polarizada. </li><li> Nós adquirimos uma imagem de estado estacionário de infravermelho com um midwave Merlin (3-5 mm) câmera infravermelha mostrado na Fig.1a, c. </li><li> Nós aplicamos uma corrente de ar frio para a área da pele do paciente contendo a lesão, bem como uma região de 50 mm de diâmetro ao redor para a duração de um minuto. </li><li> Após um minuto, nós removemos esse estresse de resfriamento para permitir que a pele para re-aquecer em temperatura ambiente dentro de 3-4 minutos (fase de recuperação térmica) (Fig. 1c-d). </li><li> Durante a fase de recuperação térmica, imagens infravermelhas da lesão pigmentada são capturadas a cada 2 segundos (Fig. 1c-d). </li><li> Todas as imagens IR (além da luz branca e luz polarizada imagens) tomado durante o estudo são gravadas e armazenadas utilizando o software Labview. </li></ol> 3. Processamento de Imagem <ol><li> As imagens IR são analisados ​​usando um código Matlab dedicada, a fim de obter resultados precisos distribuições de temperatura transiente na superfície da pele. Para este fim, apresentamos vários passos de calibração e um sistema de análise multimodal da imagem. </li><li> Começamos com a aplicação de um algoritmo de detecção de marco para a imagem de luz brilhante para localizar os cantos do marcador adesivo. Em seguida, identificamos os pontos correspondentes na imagem IR de referência. </li><li> , A fim de compensar o movimento do corpo / membros involuntários do paciente, nós usamos esses pontos como pontos de referência em um modelo quadrático de movimento para alinhar a seqüência de imagens IR durante a fase de recuperação. </li><li> Usamos o caminhante aleatório, um algoritmo de segmentação interativa de imagens onde o usuário pode espacialmente orientar a segmentação, colocando pontos de semente, para criar uma imagem de máscara delinear a lesão. </li><li> Uma vez que determinam a forma da lesão, identificamos a região correspondente em cada uma das imagens registradas IR. </li><li> Nós selecionamos pontos aleatórios no interior da lesão e longe da lesão que representa a lesão eo tecido saudável, respectivamente. </li><li> Nós comparamos a resposta transiente térmico da pele saudável e da resposta da lesão. </li><li> Preparamos uma tabela mostrando todos os dados: digital, dermatoscopia, codificados por cores imagens IR da lesão e área circunvizinha gravado em condições ambientais e 2 segundos após a excitação de arrefecimento, ea resposta transiente térmico da lesão e os tecidos saudáveis. </li></ol> 4. Resultados representativos: <img src="/files/ftp_upload/2679/2679fig1.jpg" alt="Figura 1" /> Figura 1. a) A fotografia do HRIS imagem infravermelha do sistema na sala de ensaio clínico, b) da maior área de superfície corporal com um cluster de lesões pigmentadas e armação modelo aplicado para imagens, c) imagem de referência da região de infravermelho à temperatura ambiente, d) mesma área após o resfriamento e e) seção ampliada da lesão melanoma e arredores <img src="/files/ftp_upload/2679/2679fig2.jpg" alt="Figura 2" /> Figura 2. Sala de exames com o nosso sistema de imagem térmica. <img src="/files/ftp_upload/2679/2679fig3.jpg" alt="Figura 3" /> Figura 3. Cooling da lesão e do tecido circunvizinho da pele soprando uma corrente de ar frio a partir de um 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>

Não há conflitos de interesse declarados.

Os resultados sugerem que o estresse através da aplicação de refrigeração que melhor as diferenças de temperatura entre a lesão eo tecido saudável circundante. Além disso, por causa de pequenos movimentos da paciente durante a geração de imagens térmicas, tivemos de aplicar movimento de monitoramento corretamente sobreposição de imagens para medir diferenças de temperatura entre o estado de referência e a distribuição de temperatura durante a recuperação térmica. Sem o acompanhamento do movimento, não teríamos sido capazes de detectar e medir a diferença de temperatura entre a lesão maligna e os tecidos saudáveis. Estes resultados ea necessidade de movimento precisa de acompanhamento explicam os investigadores dificuldades enfrentadas no passado, quando a tentativa de diagnosticar melanoma utilizando IR de imagem com base em informações de estado estacionário só e provam claramente as vantagens da termografia dinâmica. Note-se que a resolução espacial da câmera IR (número de pixels na matriz plano focal IR) é crítico quando discernir pequenas lesões. Tanto a resolução espacial ea sensibilidade da temperatura de início das câmeras de infravermelho foi limitado, o que também contribui para as dificuldades na detecção de melanoma estágio inicial no passado. As principais diferenças entre nossa abordagem e as tentativas anteriores de imagem térmica - que foram moderadamente bem sucedida - são as seqüências de calibração e as etapas de processamento de imagem, que nos permitem medir com precisão as diferenças de temperatura nesse sistema, além do processo de imagem dinâmica que se baseia em arrefecimento activo.

<table border="1"><tbody><tr><td> Nome do equipamento </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Merlin MWIR câmera </td><td> FLIR </td><td> - </td></tr><tr><td> Canon PowerShot G11 </td><td> Cânone </td><td> - </td></tr><tr><td> DermLite Foto Sistema </td><td> DermLite </td><td> - </td></tr><tr><td> Tubo de vórtice </td><td> EXAIR </td><td> - </td></tr><tr><td> Tanque de ar </td><td> Airgas </td><td> - </td></tr></tbody></table>

quantitative visualization and detection of skin cancer using dynamic thermal imaging

Em 2010 cerca de 68.720 melanomas serão diagnosticados somente nos EUA, com cerca de 8.650 resultando em morte 1. Até o momento, o único tratamento eficaz para o melanoma continua a excisão cirúrgica, portanto, a chave para a sobrevivência prolongada é a detecção precoce 2,3. Considerando o grande número de pacientes diagnosticados a cada ano e as limitações no acesso a cuidados especializados de forma rápida, objetiva o desenvolvimento de instrumentos de diagnóstico in vivo para auxiliar o diagnóstico é essencial. Novas técnicas para detectar o câncer de pele, especialmente não-invasivo ferramentas de diagnóstico, estão sendo exploradas em numerosos laboratórios. Junto com o cirúrgico métodos, técnicas como a fotografia digital, dermatoscopia, sistemas de imagens multiespectrais (MelaFind), sistemas de laser (microscopia confocal a laser de varredura a laser de imagens de perfusão doppler, tomografia de coerência óptica), ultra-som, ressonância magnética, estão sendo testados . Cada técnica oferece vantagens e desvantagens, muitos dos quais representam um compromisso entre eficácia e precisão versus facilidade de uso e considerações de custo. Detalhes sobre estas técnicas e as comparações estão disponíveis na literatura 4. Infrared imaging (IR) mostrou-se um método útil para diagnosticar os sinais de determinadas doenças através da medição da temperatura da pele local. Há um grande corpo de evidências mostrando que a doença ou desvio do seu funcionamento normal são acompanhadas por mudanças da temperatura do corpo, que por sua vez afeta a temperatura da pele 5,6. Dados precisos sobre a temperatura do corpo humano e da pele pode fornecer uma riqueza de informações sobre os processos responsáveis ​​pela geração de calor e termorregulação, em particular o desvio das condições normais, muitas vezes causada por doença. No entanto, IR de imagem não tem sido amplamente reconhecida na medicina devido ao uso precoce da tecnologia 7,8 várias décadas atrás, quando a precisão de medição de temperatura e resolução espacial foram ferramentas de processamento de imagem inadequada e sofisticado não estavam disponíveis. Esta situação mudou drasticamente no final dos anos 1990s-2000s. Avanços na instrumentação IR, a implementação de algoritmos de processamento digital de imagem e de imagem IR dinâmica, que permite aos cientistas analisar não só o espaço, mas também o comportamento temporal térmica da pele 9, permitiu avanços no campo. Em nossa pesquisa, explorar a viabilidade de IR de imagem, combinada com estudos teóricos e experimentais, como um custo eficaz, não invasivo, de técnica de medição óptico para detecção in vivo de tumores, com ênfase sobre o rastreamento e detecção precoce do melanoma 13/10 . Neste estudo, mostramos os dados obtidos em um estudo clínico em que pacientes que possuem uma lesão pigmentada com uma indicação clínica para biópsia são selecionados para a imagem latente. Nós comparamos a diferença nas respostas térmica entre tecidos saudáveis ​​e malignas e compararam os dados com os resultados da biópsia. Concluímos que o aumento da atividade metabólica da lesão melanoma pode ser detectado por imagem infravermelha dinâmico.

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

Nós demonstramos que as lesões malignas pigmentadas com uma maior atividade metabólica gerar quantidades de calor e quantificáveis ​​a medição da resposta transiente térmico da pele para uma excitação de refrigeração permite a identificação quantitativa de melanoma e outros cânceres de pele (vs. não-proliferativa nevos), numa fase inicial estágio da doença.

Visualization quantitativos e Detecção de Câncer de Pele Usando imagens térmicas dinâmico

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 ...

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16.6K visualizações.  The Johns Hopkins University. Nós demonstramos que as lesões malignas pigmentadas com uma maior atividade metabólica gerar quantidades de calor e quantificáveis ​​a medição da resposta transiente térmico da pele para uma excitação de refrigeração permite a identificação quantitativa de melanoma e outros cânceres de pele (vs. não-proliferativa nevos), numa fase inicial estágio da doença.

Vídeo: 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.

Medição precisa e simples do pró-inflamatórias IL-1β citocinas usando um ensaio Estimulação Sangue Total

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

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.

Análise quantitativa de metástase de câncer utilizando um modelo de Embrião aviária

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.

Luminescência de Cerenkov Imaging (CLI) para o Monitoramento Terapia de Câncer

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.

Análise Quantitativa Multispectral Após transplante de tecido fluorescente para visualização das Origens celulares, Tipos e Interações

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 cefalorraquidiano MicroRNA perfil usando Quantitative PCR em Tempo Real

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.

Medição da dinâmica Escapulário Cinemática Usando um Cluster acrômio marcador para Minimizar Artefato Movimento Pele

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.

Contraste Dinâmico aprimorada Ressonância Magnética de um Modelo de câncer de pâncreas Orthotopic Rato

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.

Difusa espectroscopia óptica para a avaliação quantitativa da Aguda de Radiação Ionizante toxicidade cutânea induzida pelo uso de um modelo de rato

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.

Visualização Quantitativa de Infiltrado de Leucócitos em um Modelo Murino de Miocardite Fulminante por Microscopia de Folha de Luz

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.

Análise quantitativa [18F]-NAF-PET-RM para avaliação da rotatividade óssea dinâmica em paciente com dor nas costas Facetogênica

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