Este estudo foi apoiado por CCSRI Grant # 700537 e Grant CIHR # 84535 para JDL e NIH / NCI conceder # CA120711-01A1-01A1 e CA120711 a AZ. Todos os experimentos foram realizados em conformidade com as normas e diretrizes do Animal Care Use Institucional e Comitê da Universidade da Califórnia em San Diego, Animal Care e Uso da Universidade de Western Ontario.

1. Inoculação do tumor em CAM embrião aviária <ol><li> Prepare shell-less embriões de galinha fertilizados, conforme descrito 8. </li><li> No dia 9 do desenvolvimento embrionário, montar uma micro-injector utilizando uma agulha de calibre 18 conectada em uma seringa mL. Corte um pedaço 5-6 polegadas de tubo Tygon (1 / 32 &quot;de diâmetro interior, 3 / 32&quot; de diâmetro externo, 1 / 32 &quot;a espessura da parede) e insira cuidadosamente bisel da agulha na tubulação. Aproximadamente 4-5 polegadas da tubulação deve estendem desde a ponta da agulha (Figura 1a). </li><li> Encha a seringa e tubo com suspensão de células. Em seguida, insira uma agulha de vidro microinjeção no final da tubulação e remova cuidadosamente as bolhas de ar. </li><li> Injetar Dia 9 embriões (Figura 1b), sob um escopo dissecção com um iluminador com 10.000-100.000 células do câncer como um bolo dentro do CAM (Figura 1c). Lentamente injetar células e cuidadosamente garantir que a agulha está no lugar correto para as células para formar um bolo visível dentro da CAM. Células que por gotejamento sobre a superfície CAM podem ser limpos utilizando aplicador Kimwipe ou outros. </li><li> Retorno embriões para incubadora umidificada a 38 ° C com umidade de 60% e permitir que o tumor cresça e irrigar (até 7 dias). </li></ol> 2. Preparação de nanopartículas <ol><li> Para preparar nanopartículas fluorescente etiquetado CPMV para injecção no embrião de galinha; diluir as nanopartículas viral (sintetizado como 8 em PBS, pH 7,4, a uma concentração de 100 mg / ml mistura Vortex bem antes de usar e centrifugar por um minuto para remover qualquer. agregados Sonication. também pode ser útil dependendo conjugados e grau de agregação. Stocks de CPMVs fluorescente etiquetado são estáveis ​​por pelo menos 1 ano quando armazenado a 4 ° C no escuro. Verifique periodicamente ações, colocando algumas gotas numa lâmina de vidro e verificação de fluorescência. microscopia eletrônica de transmissão (TEM) também pode ser usado para determinar se as partículas permaneceram intactas após conjugação. </li><li> Resumidamente, um micrograma de nanopartículas viral (em um volume final de 2 mL) podem ser adicionados às redes de cobre revestido. Em seguida, adicione um volume igual de acetato de uranila 2% durante 1 minuto, microscópio eletrônico (Philips EM 410). </li></ol> 3. Injeção intravenosa de fluorescência marcado com nanopartículas viral <ol><li> No dia 16, montar micro-injector, como descrito acima. Elaborar volume desejado de nanopartículas viral através do tubo para dentro da seringa (&gt; mL 200). Remova cuidadosamente as bolhas de ar. Inserir a agulha de vidro microinjeção no final da tubulação. Assegurar que a agulha é tão longo e afilado possível (Figura 2a). Se a agulha estiver muito fechado, não será capaz de perfurar o ectoderma e deixará de penetrar no navio. Se a agulha estiver muito acentuado, a ponta entrará em colapso ao penetrar tecidos. </li><li> Por via intravenosa injetar 100 ml de 1 mg / ml de fluoresceína dextran (70.000 MW Da, Invitrogen) em embrião tendo tumor distal do site desejado a ser visualizado (Figura 3d). Canulação da veia CAM sucesso é evidente pela clearing de sangue no caminho do fluxo da injeção (Figura 2b). Avaliar vascular 1 hora após a injeção de vazamento. </li><li> Por via intravenosa injetar 50 ul de 1 mg / ml de solução de CPMV-Alexa Fluor 647 (CPMV AF-647) ou peguilado CPMV-Alexa Fluor 647 (CPMV-PEG-AF 647) em embriões com tumores com vazamento vascular distal do site desejado ser visualizados. </li><li> Tumores imagem imediatamente ea cada hora após a injeção usando uma Zeiss Examiner Z1 microscópio vertical equipado com um disco giratório Yokogawa, Hamamatsu Imagem 9100-12 câmera EM-CCD. </li></ol> 4. Em tempo real de imagens intravital <ol><li> Para montar a unidade de imagem do embrião, aplique uma fina camada de graxa de vácuo em torno da circunferência do porto de imagem na parte inferior da tampa da unidade de imagem do embrião, e se encaixam uma lamela de vidro 18 milímetros para o porto. </li><li> Posição do embrião de tal forma que a lamela vai cobrir a área desejada para a imagem latente. Lentamente abaixe a tampa até que a lamínula só faz contato com o embrião, e então parafuso a tampa sobre a unidade para segurá-la no lugar (Figura 2c). </li><li> Adicione a água aquecida a 37 ° C para a unidade de imagem do embrião fora do prato contendo o embrião, e em seguida, coloque a unidade inteira para o palco de um microscópio confocal na vertical com o interior da câmara ambiental equilibrado a 37 ° C. </li><li> Unidade de posição da imagem contendo o embrião sob o Spinning microscópio de fluorescência confocal de disco (Figura 2e). A unidade de imagem irá realizar o embrião no lugar e manter o campo de visão, enquanto a captura de imagens fixas, permitindo a ambas as pilhas tridimensionais Z e lapso de tempo que as imagens sejam capturadas. Nós adquirir e analisar tridimensional lapso de tempo imagens usando Perkin Elmer (anteriormente Improvisation) Pacote de software Volocity (Figura 3a). Adquirir uma alta resolução tridimensional pilha do tumor e vascularização em torno de visualizar detailed análises estruturais específicas em tempo de pontos. Adquirir tridimensional pilhas em intervalos regulares de tempo, aponta para mapa detalhado mudanças estruturais na vasculatura tumoral. Tumores de imagem a cada hora após a injeção. </li><li> Quantificar a absorção de nanopartículas viral através do cálculo da média Alexa Fluor (AF) 647 sinal dentro de regiões selecionadas no tumor ou no estroma software quantificação (não-tumorais área) usando a imagem como Volocity (Perkin Elmer). O tumor de estroma proporção foi calculada dividindo a média AF 647 sinal no tumor sobre a média AF 647 sinal no estroma. A relação tumor / estroma maior que 1 indica que as nanopartículas está sendo tomada pelo tumor. </li></ol> 5. Resultados representativos: No exemplo aqui descrito, injetamos HT-29 células de câncer de cólon para formar um bolo de aproximadamente 1 milímetro de tamanho dentro da CAM de embriões dia 9 de frango (Figura 1b). Após a inoculação, os embriões foram cultivados por 7 dias em uma incubadora umidificada para permitir o crescimento do tumor suficiente e vascularização (Figura 1c). Embriões foram injetados por via intravenosa com um baixo peso molecular dextran para confirmar a vascularização do tumor, e os tumores foram visualizadas ao microscópio Zeiss Z1 AxioExaminer vertical (Figura 2d). Após a administração intravenosa de CPMV AF-647 ou CPMV-PEG-AF 647 nanopartículas (Figura 3a e b), de alta resolução em tempo real confocal de imagem (Figura 2e) revelou que tanto CPMV e CPMV-PEG nanopartículas rapidamente rotulados toda a vasculatura, mas a captação de CPMV-PEG pelo tumor foi de aproximadamente 3 vezes superior CPMV após 12 horas (Figura 3). A captação tumoral relativa de nanopartículas foi determinada utilizando software de análise de imagem (Volocity da Perkin Elmer). Regiões de interesse foram selecionados dentro e fora do tumor (no compartimento estromal) ea intensidade média de fluorescência de cada um era determinado. Os dados são expressos como tumor / estroma ratio. <img src="/files/ftp_upload/2808/2808fig1.jpg" alt="Figura 1"/> Figura 1. Microinjeção de tumores no CAM de um embrião aviária. (A) O aparelho microinjeção é montado a partir dos componentes, como indicado. (B) os embriões das aves no dia 9 está pronto para ser inoculado com tumor quando o CAM tem se espalhado para cobrir toda a superfície. (C) No dia 16, o tumor terá crescido a até 1 cm de diâmetro (linha pontilhada) e está pronto para a injecção com nanopartículas. <img src="/files/ftp_upload/2808/2808fig2.jpg" alt="Figura 2"/> Figura 2. Injeção de nanopartículas e imagem intravital. Injeção sob um microscópio de dissecação mostrando (a) a ponta da agulha microinjetor pronto para ser injetado na veia CAM e (b) a agulha microinjetor introduzida na veia (indicado pela seta) e nanopartículas injetadas no fluxo de sangue (visto limpando de sangue). (C) da unidade de imagem contendo embrião aviária com a lamela interface diretamente com o CAM. (D) Antes da injeção de nanopartículas, vascularização do tumor é avaliada através de imagens intravital após a injeção de fluoresceína dextran. (E) da unidade de imagem contendo o embrião posicionado no palco de um microscópio confocal na vertical dentro de uma temperatura regulada a clausura a 37 ° C. <img src="/files/ftp_upload/2808/2808fig3.jpg" alt="Figura 3"/> Figura 3. Visualização intravital da captação de nanopartículas em tumores humanos. Tumores são visualizados sete horas após a injeção de (a) CPMV-AF647 e (b) CPMV-PEG-AF647. d) Excisão de tumor de embrião de aves para análise posterior.

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A membrana corioalantóide (CAM) do embrião aviária é um modelo útil para avaliar a dinâmica vascular e farmacocinética de tumores humanos. A estrutura ea posição do CAM permite a aquisição de imagem de alta qualidade e acomoda de muitos tipos de câncer xenoenxertos sem procedimentos cirúrgicos invasivos. Além disso, xenotransplantes tumorais do câncer implantados na membrana corioalantóide tornar vascularizado dentro de 7 dias, oferecendo um meio rápido, barato e semi-high-throughput para avaliar o acúmulo de nanopartículas no tecido tumoral. Desde xenoenxertos câncer implantado no CAM de embriões shell-less de frango são acessíveis para as ópticas de alta resolução de um microscópio de epifluorescência, na posição vertical ou confocal, a informação contextual e temporal em relação a captação de nanopartículas na vasculatura do tumor pode ser facilmente obtida. Xenoenxerto de câncer neste modelo tendem a crescer lateralmente através da CAM, resultando em tumores que são grandes, permanecendo menos de 200 m de profundidade. Isso os torna particularmente adequado para geração de imagens intravital por causa de epifluorescência microscópios padrão pode penetrar eficazmente a massa do tumor inteiro. Em contraste, os tumores implantados em ambos os sítios superficiais ou ortotópico dentro do rato proliferam em três dimensões, o que torna difícil localizar com precisão as nanopartículas profunda dentro desses tumores por técnicas não invasivas. Nós utilizamos este modelo para avaliar a aceitação de pontos quânticos, lipossomas, nanopartículas de óxido de ferro e em um número de xenotransplantes tumorais humanos, destacando o potencial para este modelo a ser apropriado para o análise in vivo de uma ampla gama de formulações de nanopartículas.

<table border="1"><tbody><tr><th> Nome reagente / Equipamentos </th><th> Companhia </th><th> Número Catálogo </th><th> Comentários </th></tr><tr><td> Ovos fertilizados leghorn </td><td> Frey `s Hatchery, St. Jacobs </td><td> N / A </td><td></td></tr><tr><td> Ferramenta rotativa Dremel </td><td> Dremel </td><td></td><td> Pode ser usado qualquer modelo </td></tr><tr><td> Rodas de corte Dremel não. 36 </td><td> Dremel </td><td> 409 </td><td></td></tr><tr><td> Sportsman nascedouro </td><td> Berry Hill </td><td> 1550HA </td><td></td></tr><tr><td> Sportsman incubadora </td><td> Berry Hill </td><td> 1502EA </td><td></td></tr><tr><td> Etanol </td><td></td><td></td><td> 70% (vol / vol) </td></tr><tr><td> Poliestireno pesar barcos </td><td> VWR </td><td> 12577-01 </td><td></td></tr><tr><td> Pratos quadrados Petri </td><td> Simport, VWR </td><td> 25378-115 </td><td></td></tr><tr><td> Rubbermaid recipiente com tampa de borracha </td><td> Guillevin </td><td> RH3-228-00-BLU </td><td> furos em lados </td></tr><tr><td> Vertical pipeta extrator </td><td> David Kopf Instruments </td><td> modelo 720 </td><td> Settings: 16,3 (aquecedor) e 2,3 (solenóide) </td></tr><tr><td> Sódio tubos de vidro de borosilicato capilar </td><td> Instrumento Sutter </td><td> BF100-58-10 </td><td> (OD, 1,0 mm; ID 0,58 milímetros; de 10 cm comprimento) </td></tr><tr><td> Modificado 1X Dulbecco Eagle Medium (DMEM) </td><td> Invitrogen </td><td> 11995073 </td><td></td></tr><tr><td> Dulbecco </td><td> Invitrogen </td><td> 14190250 </td><td> pH 7,4 </td></tr><tr><td> Tampão fosfato (D-PBS) (1X), líquido </td><td></td><td></td><td></td></tr><tr><td> Tripsina, 0,05% (1X) com EDTA 4NA, líquido </td><td> Invitrogen </td><td> 25300054 </td><td></td></tr><tr><td> Soro fetal bovino </td><td> Invitrogen </td><td> 12483-020 </td><td> Calor inativar </td></tr><tr><td> Hemocitômetro </td><td> Hausser Scientific, VWR </td><td> 15170-090 </td><td></td></tr><tr><td> Centrifugador </td><td> Eppendorf modelo 5810R </td><td> 5811 000.010 </td><td></td></tr><tr><td> De ponta fina forceps </td><td> VWR </td><td> 25607-856 </td><td></td></tr><tr><td> Tygon R-3603 tubulação </td><td> VWR </td><td> 63009-983 </td><td> 50 pés (diâmetro interno 1/32-inch, diâmetro externo 3/32-inch, espessura da parede 1/32-inch </td></tr><tr><td> Agulhas hipodérmicas para injectáveis ​​calibre 18 agulhas </td><td> BD </td><td> 305195 </td><td> Caixa de 100 </td></tr><tr><td> 1 ml seringas para injectáveis </td><td> BD </td><td> 309602 </td><td> Caixa de 100 </td></tr><tr><td> Iluminador de fibra óptica de microscópio </td><td> Amscope </td><td> HL250-AY </td><td> 150W </td></tr><tr><td> kimwipes </td><td> VWR </td><td> 10805-905 </td><td></td></tr><tr><td> Sementes de V. unguiculata (California preto olho-no. 5) </td><td> Burpee </td><td> 51771A </td><td></td></tr><tr><td> Indoor luzes crescimento </td><td> SunLite, Abastecimento jardineiro </td><td></td><td></td></tr><tr><td> Metil-éster PEO4-NHS </td><td> Perfurar </td><td> PI22341 </td><td></td></tr><tr><td> MPEG-NHS, PEG succinimidyl éster, MW 2000 </td><td> NANOCS </td><td> PEG1-0002 </td><td></td></tr><tr><td> Alexa Fluor 647 ácido carboxílico (succinimidyl éster) </td><td> Invitrogen </td><td> A20006 </td><td></td></tr><tr><td> Oregon Verde 488 éster succinimidyl * 6-isômero * </td><td> Invitrogen </td><td> O-6149 </td><td></td></tr><tr><td> Dimetilsulfóxido (DMSO) </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Dibásico monohydrogen fosfato </td><td> Sigma </td><td> 379980 </td><td> K2HPO4 (para buffer phopshate) </td></tr><tr><td> Monobásico dihidrogenofosfato </td><td></td><td> P5655 </td><td> KH2PO4 (para buffer phopshate) </td></tr><tr><td> Superose 6 coluna de tamanho-exclusão </td><td> GE Healthcare ciências da vida </td><td> 17-0673-01 </td><td></td></tr><tr><td> AKTA Explorador 100 Cromatógrafo </td><td> GE Amersham Pharmacia </td><td> WS-AKTA100 </td><td></td></tr><tr><td> AKTA kit de alto fluxo </td><td> GE Healthcare ciências da vida </td><td> 18-1154-85 </td><td></td></tr><tr><td> Sacarose </td><td> Sigma </td><td> S0389 </td><td></td></tr><tr><td> Ultracentrífuga </td&gt; <td> Beckman </td><td></td><td></td></tr><tr><td> SW 28 Ti rotor </td><td> Beckman </td><td> 342204 </td><td> Balde balanço </td></tr><tr><td> 50,2 Ti rotor </td><td> Beckman </td><td> 337901 </td><td> Ângulo fixo </td></tr><tr><td> Amicon Ultra-15 Centrífuga Unidades Filtro </td><td> Millipore </td><td> UFC910008 </td><td> 100 kDa cortado </td></tr><tr><td> Gradiente de ex- </td><td> Biorad </td><td></td><td></td></tr><tr><td> dextran, fluoresceína, 70.000 MW, aniônicos </td><td> Invitrogen </td><td> D1823 </td><td></td></tr><tr><td> Spinning microscópio de fluorescência confocal de disco </td><td> Quorum; Yokogawa CSU 10, Yokogawa </td><td> N / A </td><td></td></tr><tr><td> Microscópio de campo amplo de epifluorescência </td><td> Quorum; Zeiss Axio Examiner, Zeiss </td><td> N / A </td><td></td></tr><tr><td> Hamamatsu Imagem 9100-12 câmera EM-CCD </td><td> Quorum; Hamamatsu </td><td> N / A </td><td></td></tr><tr><td> Unidade de temperatura do compartimento para microscópio </td><td> Plásticos de Precisão </td><td> N / A </td><td></td></tr><tr><td> Graxa de vácuo </td><td> VWR </td><td> 59344-055 </td><td></td></tr><tr><td> Lamínulas de vidro circulares não. 1 (18 mm) </td><td> VWR </td><td> 16004-300 </td><td></td></tr><tr><td> Software Volocity </td><td> Perkin Elmer </td><td></td><td></td></tr><tr><td> Pinto gabinete embrião </td><td></td><td> fabricados sob medida </td><td></td></tr><tr><td> Tesoura delicada </td><td> VWR </td><td> 25608-203 </td><td></td></tr><tr><td> Formalina </td><td> Bioshop </td><td> FOR201.500 </td><td> Uso em fumehood </td></tr><tr><td> Corte ideal </td><td> Fisher; Tissue Tek </td><td> 1437365 </td><td></td></tr><tr><td> Temperatura (OCT) </td><td></td><td></td><td></td></tr><tr><td> Moldes de plástico </td><td> Pescador </td><td> 22-038217 </td><td></td></tr><tr><td> VWR VistaVision HistoBond Adhesive Slides </td><td> VWR </td><td> 16004-406 </td><td></td></tr><tr><td> Prolongar a ouro com DAPI </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td> Lâminas descartáveis ​​para criostato </td><td> Pescador </td><td> 12-634-2 </td><td></td></tr><tr><td> Criostato </td><td> Leica CM 3050 S </td><td> 14047033518 </td><td></td></tr></table>

evaluation of nanoparticle uptake in tumors in real time using intravital imaging

As tecnologias atuais para a imagem latente do tumor, como ultra-som, ressonância magnética, PET e CT, são incapazes de produzir imagens de alta resolução para a avaliação da absorção de nanopartículas em tumores em nível microscópico 1,2,3, destacando a utilidade de um modelo adequado xenoenxerto em que para realizar análises detalhadas captação. Aqui, usamos imagens de alta resolução intravital para avaliar a absorção de nanopartículas em humanos xenoenxertos tumor em um modelo de embriões modificados, shell-less frango. O modelo de embrião de galinha é particularmente bem adequada para esses análise in vivo porque ele suporta o crescimento de tumores humanos, é relativamente barato e não requer cirurgia anestesiados ou 4,5. Células tumorais forma xenoenxertos totalmente vascularizado dentro de 7 dias quando implantados na membrana corioalantóide (CAM) 6. Os tumores resultantes são visualizadas por não-invasivos de imagem em tempo real, de alta resolução que pode ser mantido por até 72 horas, com pouco impacto sobre o host ou sistemas de tumor. Nanopartículas com uma ampla gama de tamanhos e formulações administradas distal ao tumor podem ser visualizados e quantificados como elas fluem através da corrente sanguínea, extravasamento de vascularização tumoral com vazamentos, e se acumulam no local do tumor. Descrevemos aqui a análise de nanopartículas de derivados do vírus do mosaico do caupi (CPMV) decorado com infravermelho próximo corantes fluorescentes e / ou polímeros de polietileno glicol (PEG) 7, 8, 9,10,11. Após a administração intravenosa, essas nanopartículas virais são rapidamente internalizados pelas células endoteliais, resultando em rotulagem global da vasculatura tanto fora como dentro do tumor 7,12. PEGylation das nanopartículas viral aumenta sua meia-vida plasmática, estende seu tempo na circulação, e, finalmente, aumenta a sua acumulação nos tumores através da permeabilidade aumentada e efeito de retenção (EPR) 7, 10,11. A taxa ea extensão da acumulação de nanopartículas em um tumor é medido ao longo do tempo usando o software de análise de imagem. Esta técnica fornece um método para tanto visualizar e quantificar a dinâmica de nanopartículas em tumores humanos.

Watch this Scientific Journal Video about Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging at JoVE.com

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

Nós apresentamos uma nova abordagem para quantificar a localização das nanopartículas na vasculatura de tumores humanos xenografted utilizando dinâmicas, imagens em tempo real intravital em um modelo embrião aviária.

Avaliação de Captação de nanopartículas em tumores em tempo real usando imagens intravital

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

evaluation-nanoparticle-uptake-tumors-real-time-using-intravital

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JoVE Journal

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18.1K visualizações.  University of Western Ontario. Nós apresentamos uma nova abordagem para quantificar a localização das nanopartículas na vasculatura de tumores humanos xenografted utilizando dinâmicas, imagens em tempo real intravital em um modelo embrião aviária.

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

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

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 excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Imagens em tempo real multiespectrais de fluorescência para detecção intra-operatória do linfonodo sentinela em Oncologia 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 ...

Medicina

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.

Detecção MicroRNA em tumores de próstata por Quantitative PCR em tempo real (qPCR)

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

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Movimento Reciclagem com feedback em tempo real do desempenho

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imagem Óptico de tumores cerebrais e artrite Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

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.

Induzida pela quimioterapia Vascular Toxicidade - em tempo real<em&gt; In vivo</em&gt; Imagem de Impairment Vessel

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

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

Imagens de raios-X em tempo real de pulmão volumes de fluido em Neonatal mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Avaliação de metástases pulmonares em Rato mamárias tumorais Models por Quantitative PCR em tempo real

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

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.

Estendida Time-lapse Imagem intravital do Real-time multicelular Dynamics no microambiente tumoral

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

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 ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Amostragem e Extração de Tumores Cerebrais no local para análise metabolômica e lipidômica

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...