Os autores gostariam de agradecer a Laura Diaz, Kevin Phillips, Willa A. Hsueh, e King C. Li por seu apoio útil comentários e técnica no desenvolvimento deste método.

1. Preparação animal e tratamento a frio <ol><li> Localize e inspecione a 4 ° C sala fria que tenha sido aprovado para acomodar ratos de laboratório. </li><li> Pré-frio gaiolas dos animais durante a noite no quarto frio. As gaiolas são montadas sem alimentos e roupas, mas com uma garrafa de água. </li><li> Na manhã do dia experimental, os ratos lugar, uma a uma em cada uma das gaiolas pré-resfriada em intervalos de 30 min. Cada rato isoladamente enjaulado deve ficar no quarto frio por quase quatro horas antes de ser transportado para o laboratório de imagens. Garantir os ratos estão em jejum, mas com acesso à água. </li><li> Às 4 horas transporte, tratamento pós-frio um animal de uma vez a cada 30 min para o Laboratório de imagem. Isto pode ser conseguido por enchimento de um recipiente de isopor com gelo e a colocação de uma gaiola de alojamento pré-arrefecida em cima do gelo no interior da caixa. Vagamente colocar a tampa na caixa de isopor. </li></ol> 2. Configuração Micro-PET/CT imagem de fluxo de trabalho <p class=&quot;Jove_content&quot;&gt; Neste micro-PET/CT protocolo de imagem é obtida com o Inveon Siemens Dedicado PET (dPET) Sistema e Multimodalidade Inveon (MM) do sistema (CT / SPECT) no modo acoplado. O animal é colocado na entrada da MM, em primeiro lugar digitalizados com a CT para referências anatómicas, e, em seguida, deslizar para o centro do dPET para uma aquisição de PET estático F18. A fim de permitir que o computador host para realizar estas tarefas seqüenciais automaticamente, o &quot;fluxo de trabalho&quot; seguinte é programado com o local de trabalho de Aquisição Inveon software (IAW) antes da sessão de imagem real. <ol><li> Aquisição CT: Para um corpo inteiro TC, definida como a atual em 500 uA, tensão em 80kV, tempo de exposição a 200 ms e 240 passos para 240 ° de rotação. Para detector de raios-X, selecione a resolução de &quot;ampliação de sistema de baixo&quot; com 78 mm axial campo de imagem e modo de cama de solteiro. Selecione &quot;reconstrução tempo real&quot; com o &quot;Common Cone Beam-Reconstrução&quot; método para que as negociações de acolhimento de PC comum computador dedicado reconstrução em tempo real (Cobra) para iniciar a tarefa. </li><li> PET Aquisição de emissão: Conjunto de 600 segundos (10 minutos) para &quot;tempo de varredura fixa&quot; em &quot;adquirir pelo tempo&quot; opção. Selecione F-18 como &quot;estudo de isótopos&quot; e usar 350-650 keV como &quot;nível de energia&quot;. </li><li> PET Histograma de emissão: &quot;quadro dinâmico&quot; Definir como &quot;negro&quot; para processar os dados como uma moldura para toda a duração de alcançar varredura estática. Selecione &quot;3D&quot; como o tipo de histograma e escolher &quot;correção sem dispersão&quot;. </li><li> PET Reconstrução: Use Maximização da Expectativa 2D ordered subset (OSEM2D) como o algoritmo de reconstrução. </li></ol> 3. A injeção de FDG <ol><li> Encomendar um pacote clínico de 18 F-FDG (10 mCi) a partir de um fornecedor regional para a sua chegada ao laboratório de imagens ~ 30 min antes da administração programada primeiro. Siga os procedimentos do instituto de segurança para receber e examinar o pacote contendo materia radioativols (RAM). </li><li> Com a proteção fornecida por um escudo topo L-bloco de mesa, alíquota do FDG e fazer diluições com solução salina estéril. A concentração da atividade diluída de FDG deve estar disponível em 200-300 ul μCi/100 para cada injeção. Extrair a solução de FDG para uma seringa de 1 ml com 26G meia polegada da agulha e medir a radioactividade da seringa inteiro com um calibrador de dose. </li><li> Injectar o animal que está a apenas transportados da sala fria (ver Passo 1.4) com 100 ul de solução de FDG através da via intraperitoneal (ip). Registre o tempo de injeção. Medir a radioactividade do resíduo seringa novamente com o calibrador de dose. </li><li> Colocar o animal de volta para a jaula frio dentro de um refrigerador de isopor com gelo mantido. Incubar o animal no frio (~ 4 ° C) durante 1 hora para absorção de FDG. </li><li> Calcular a actividade da FDG injectado para cada ratinho através da seguinte fórmula: Injetado atividade (iCi) = atividade na seringa antes da injeção- Atividade em seringa após a injeção </li></ol> 4. Micro-PET/CT imagem <ol><li> A imagiologia micro-PET/CT começa 1 hora após a injecção de FDG ou 5 horas após o tratamento a frio. Coloque o animal em uma câmara de indução anestésica com 3% de isoflurano em oxigênio. </li><li> Uma vez que a anestesia é induzida, o animal é movido para uma pastilha de micro-CT (cama de animais) com a sua cabeça apoiada no interior de uma máscara facial de cone que proporciona continuamente isoflurano (2%) a uma taxa de fluxo de 2 L / min. Uma almofada de aquecimento eléctrico (Biovet, m2m Imaging Corp) é colocado sob o animal para ajudar a manter a temperatura do corpo. </li><li> Deslize o animal até a entrada do scanner MM, ativar o &quot;laser&quot; ícone na barra de ferramentas, e usar o controle touchpad para mover a cama de modo que o peito do animal é a cruz de linhas de laser horizontal e vertical. Na janela &quot;Laser Alinhar&quot;, selecione &quot;tipo de digitalização primeira&quot;, como tomografia computadorizada, e &quot;aquisição PET incluído no fluxo de trabalho&quot;, como o option. </li><li> Abra o &quot;Scout View&quot; janela e adquirir uma visão batedor de raios-x radiografia. Use IAW para ajustar a posição da cama dos animais de modo que o centro do campo de vista da CT está localizado no centro do corpo do rato (fígado). Repita este passo, se necessário. </li><li> Inicie o &quot;fluxo de trabalho&quot;, estabelecido no Passo 2. Quando as opções de pop-up, selecione uma adequada 3D PET-CT transformação arquivo de matriz para ser usado no CT de reconstrução, e escolher um arquivo de normalização recentemente criado para PET reconstrução sem correção de atenuação. O IAW, então, começar CT e PET sequencialmente como programado. </li><li> Depois de todo o fluxo de trabalho é completado, o que pode demorar 20-25 min, rapidamente avaliar a qualidade da CT adquirido e as imagens de PET com o VM ASIPro, um software de análise de micro-PET co-instalado com MDI. Arquivar os dados de imagem em um dispositivo de armazenamento de dados ou transferir os dados através da rede para uma estação de trabalho de pós imagem análise (ver Etapa 5) para análise posterior. </li><li> Release o animal a partir dos sistemas de imagem e colocá-lo a uma gaiola de habitação limpo com comida normal e o abastecimento de água para a sua recuperação, à temperatura ambiente. Os sistemas são agora pronto para que o animal seguinte na fila. Observe o cuidado e manuseio de pós-imagem animais devem seguir as normas do instituto sobre &quot;manipulação de animais de laboratório injetados com RAM&quot;. Observe também que as agulhas usadas / seringas, os absorventes, luvas e roupa de cama todos e matéria fecal devem ser considerados como resíduos radioactivos, e tratadas de acordo com relevantes do instituto RAM regulamentos de eliminação de resíduos. </li></ol> 5. Pós-imagem Análise <ol><li> Aberto Pesquisa Inveon Workplace software (IRW) (Siemens) e importar manualmente CT e conjuntos de dados de PET. Co-registo CT e imagens de PET em &quot;Registo&quot; janela usando ferramentas com o &quot;General análise&quot;, função e sob a &quot;Revisão&quot; janela garantir um perfeito alinhamento entre CT e PET imagens em 3 dimensões. </li><li>De &quot;região de interesse (ROI) Quantificação&quot; janela, com as referências fornecidas pelas imagens co-registradas CT, identificar BAT na região interescapular do pescoço, o mais predominante BAT frio induzida em ratos adultos, e desenhar volume de juros (VOI) da BAT sobre o conjunto de dados PET. Selecione &quot;Intensidade Voxel&quot; como o &quot;Tipo de Quantificação&quot; e registrar a média radioatividade dentro do VOI como Bq / ml. Um factor de calibração, que converte as contagens / seg para Bq / ml foi previamente estabelecido pela digitalização de um fantasma com radioactividade conhecida. </li><li> FDG na BAT é quantificado como dose percentual injectada por grama de tecido (% ID / g) com correção de decadência. A dose injectada é o resultado do passo 3.5, no entanto, converteu-se a unidade de becquerel (Bq) (1 uCi = 37000 Bq); assumimos que 1 ml de tecido igual a 1 g. </li></ol>

<ol>
	<li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+importance+of+brown+adipose+tissue+in+energy+homeostasis.">The role and importance of brown adipose tissue in energy homeostasis.</a> Curr. Opin. Pediatr. 22, 478-484 (2010).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+function+and+physiological+significance.">Brown adipose tissue: function and physiological significance.</a> Physiol. Rev. 84, 277-359 (2004).</li><li>Collins, S., Surwit, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-adrenergic+receptors+and+the+control+of+adipose+tissue+metabolism+and+thermogenesis.">The beta-adrenergic receptors and the control of adipose tissue metabolism and thermogenesis.</a> Recent Prog. Horm. Res. 56, 309-328 (2001).</li><li>Ouellet, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outdoor+temperature,+age,+sex,+body+mass+index,+and+diabetic+status+determine+the+prevalence,+mass,+and+glucose-uptake+activity+of+18F-FDG-detected+BAT+in+humans.">Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-detected BAT in humans.</a> J. Clin. Endocrinol. Metab. 96, 1115-1125 (2011).</li><li>Cypess, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> N. Engl. J. Med. 360, 1509-1517 (2009).</li><li>Fruhbeck, G., Becerril, S., Sainz, N., Garrastachu, P., Garcia-Velloso, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAT:+a+new+target+for+human+obesity.">BAT: a new target for human obesity.</a> Trends Pharmacol. Sci. 30, 387-396 (2009).</li><li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+fat+as+a+therapy+for+obesity+and+diabetes.">Brown fat as a therapy for obesity and diabetes.</a> Curr. Opin. Endocrinol. Diabetes Obes. 17, 143-149 (2010).</li><li>Virtanen, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> N. Engl. J. Med. 360, 1518-1525 (2009).</li><li>van Marken Lichtenbelt, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> N. Engl. J. Med. 360, 1500-1508 (2009).</li><li>Cohade, C., Mourtzikos, K. A., Wahl, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="USA-Fat":+prevalence+is+related+to+ambient+outdoor+temperature-evaluation+with+18F-FDG+PET/CT.">"USA-Fat": prevalence is related to ambient outdoor temperature-evaluation with 18F-FDG PET/CT.</a> J. Nucl. Med. 44, 1267-1270 (2003).</li><li>Garcia, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+brown+fat+2-deoxy-2-[F-18]fluoro-D-glucose+uptake+by+controlling+environmental+temperature+prior+to+positron+emission+tomography+scan.">Reduction of brown fat 2-deoxy-2-[F-18]fluoro-D-glucose uptake by controlling environmental temperature prior to positron emission tomography scan.</a> Mol. Imaging Biol. 8, 24-29 (2006).</li><li>Saito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58, 1526-1531 (2009).</li><li>Agrawal, A., Nair, N., Baghel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+reduction+of+brown+fat+uptake+on+FDG+PET.">A novel approach for reduction of brown fat uptake on FDG PET.</a> Br. J. Radiol. 82, 626-631 (2009).</li><li>Soderlund, V., Larsson, S. A., Jacobsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+FDG+uptake+in+brown+adipose+tissue+in+clinical+patients+by+a+single+dose+of+propranolol.">Reduction of FDG uptake in brown adipose tissue in clinical patients by a single dose of propranolol.</a> Eur. J. Nucl. Med. Mol. Imaging. 34, 1018-1022 (2007).</li><li>Zhao, J., Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenesis+is+beta3-+but+not+beta1-adrenergically+mediated+in+rat+brown+fat+cells,+even+after+cold+acclimation.">Thermogenesis is beta3- but not beta1-adrenergically mediated in rat brown fat cells, even after cold acclimation.</a> Am. J. Physiol. 275, 2002-2011 (1998).</li><li>Rowland, D. J., Garbow, J. R., Laforest, R., Snyder, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registration+of+[18F]FDG+microPET+and+small-animal+MRI.">Registration of [18F]FDG microPET and small-animal MRI.</a> Nucl. Med. Biol. 32, 567-5672 (2005).</li></ol>

Não há conflitos de interesse declarados.

Neste estudo, um método de imagem micro-PET/CT-based tem sido desenvolvido para a detecção de actividades de MTD em ratos adultos que apenas exige um tratamento a frio e uma injecção de produtos comercialmente disponíveis 18 F-FDG. Todo o procedimento pode ser realizado em um dia após o tratamento e a sequência de imagens, que se inicia a cada 30 minutos até que todos os animais são tratados e fotografada. Sob as condições experimentais descritas, um total de 10 ratos (ou 2 grupos de 5 ratos) pode...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Micro-PET/CT Imaging System </td><td> Siemens Medical Solutions EUA, Inc. </td><td> Inveon Dedicado Sistema PET e Multimodalidade Inveon CT / SPECT System (ancorado) </td><td></td></tr><tr><td> Propranolol </td><td> Sigma </td><td> P0884 </td><td></td></tr><tr><td> R $ 37.344 </td><td> Sigma </td><td> B169 </td><td></td></tr><tr><td> 18 F-FDG </td><td> Cyclotope Inc. </td><td></td><td></td></tr><tr><td> Ratos C57BL/6J Masculino </td><td> Jackson Laboratory </td><td> 000664 </td><td> 3-4 meses de idade </td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

Tecido adiposo castanho (BAT) difere do tecido adiposo branco (WAT) pela sua localização discreta e uma cor castanho-avermelhada devido à vascularização rica e de alta densidade de mitocôndrias. BAT desempenha um papel importante no gasto de energia e de não-termogénese tremendo em mamíferos recém-nascidos, bem como os adultos 1. BAT termogénese mediada é altamente regulada pelo sistema nervoso simpático, predominantemente através do receptor adrenérgico β 2, 3. Estudos recentes têm mostrado que as atividades da BAT em ​​adultos humanos são negativamente correlacionados com índice de massa corporal (IMC) e os parâmetros de outros diabéticos 4-6. BAT tem assim sido proposto como um alvo potencial para a terapia anti-obesity/anti-diabetes focando modulação do balanço energético de 6-8. Embora vários frios desafio baseados em tomografia por emissão de pósitrons (PET) são estabelecidos métodos para a detecção de BAT humana 9-13, basicamente, não há protocolo padronizado para imagens e quantifcação da BAT em modelos animais de pequeno porte, como ratos. Aqui nós descrevemos um método de imagem robusta PET / CT para avaliação funcional da BAT em camundongos. Resumidamente, os ratinhos adultos C57BL/6J foram tratados a frio sob condições de jejum por um período de 4 horas antes de receberem uma dose de 18 F-Fluorodeoxiglicose (FDG). Os ratos foram permaneceu no frio durante uma hora após a injecção adicional FDG, e, em seguida, digitalizado por um sistema micro-PET/CT pequeno animal dedicado. As imagens de PET adquiridas foram co-registradas com as imagens de CT de referências anatômicas e analisadas para FDG na área BAT interescapular de apresentar atividade BAT. Este padrão frio-tratamento e protocolo de imagem tiver sido validado através de actividades MTD teste durante intervenções farmacológicas, por exemplo, a activação BAT suprimida pelo tratamento de β-adrenoceptor antagonista propanolol 14, 15, ou a activação BAT reforçada por β3 agonista BRL37344 16. O método described aqui pode ser aplicado para o rastreio de drogas / compostos que modulam a actividade BAT, ou para identificar genes / vias que estão envolvidas no desenvolvimento e regulação de BAT em vários estudos pré-clínicos e básicos.

Um exemplo de micro-PET/CT imagiologia de BAT rato é mostrada na Figura 1. Embora a imagiologia CT fornece informações anatómicas, a imagiologia PET codifica a distribuição e a quantidade de absorção de 18 F-FDG por todo o corpo. Estes dados de imagem podem ser vistos separadamente (1A e 1B), fundido (1C), ou demonstrado com um recurso 3D, como projeção de intensidade máxima (MIP, 1D). Com a ajuda de uma ferramenta de imagem 3D, um volume de interesse (VOI), aqui na região interes...

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Um método de imagiologia funcional do rato tecido adiposo castanho (BAT), que é descrito no frio estimulada a captação de 18F-fluorodeoxiglicose (FDG) em BAT é avaliada de forma não invasiva com um protocolo padronizado micro-PET/CT. Este método é robusto e sensível para detectar diferenças nas atividades da BAT em modelos do rato.

Imagem Funcional de gordura marrom em ratos com FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

19.0K visualizações.  The Methodist Hospital Research Institute, Houston. Um método de imagiologia funcional do rato tecido adiposo castanho (BAT), que é descrito no frio estimulada a captação de 18F-fluorodeoxiglicose (FDG) em BAT é avaliada de forma não invasiva com um protocolo padronizado micro-PET/CT. Este método é robusto e sensível para detectar diferenças nas atividades da BAT em modelos do rato.

Vídeo: Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Claro / escuro Teste de transição para Ratos

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Biologia

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Rotulagem e hESCs hMSCs com óxido de ferro nanopartículas para Não-invasiva em Rastreamento vivo com ressonância magnética

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Imagem Funcional com reforço, eyetracking e Monitoramento Fisiológico

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Labirinto elevado para Ratos

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Montagem, de carga, e alinhamento de uma célula Analytical ultracentrífuga Amostra

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

Um Método para o alternante dos membros posteriores NASA modelo tradicional de Descarga em Ratos

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Mapear as sequelas de Theta Explosão Estimulação no córtex auditivo humano com Imagem Funcional

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Imagem óptica baseada em fotodiodo para dinâmica de rede de gravação com resolução de neurônio único em invertebrados não transgênicos

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...