This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

Este estudo está de acordo com a Universidade Nacional de Singapura Institutional Animal Care e Use Committee (protocolo aprovado no. R15-0225). 1. Preparação cirúrgica do Modelo Animal <ol><li> canulações navio <ol><li> Anestesiar a ratazanas macho Sprague-Dawley (6 - 7 semanas de idade) de pesagem (203 ± 20) g com cetamina (37,5 mg / ml) e xilazina (5 mg / ml) cocktail através de injecção intraperitoneal (ip) de injecção (2 ml / kg) . Não recapitular a agulha ou removê-lo a partir da seringa, após a injecção. </li><li> Uma vez que o animal foi anestesiado (confirmado por dedo do pé que comprime a), colocá-la sobre uma almofada de aquecimento para manter a sua temperatura do corpo a 37 ° C. Delicadamente raspar o cabelo na escápula, colo do útero anterior, abdômen, perna medial e saco escrotal. Gentilmente conter as pernas usando fitas de papel adesivas. </li><li> Executar todos os procedimentos cirúrgicos com uma tesoura de microdissecção e pinças angulares durante a visualização atravésum estereomicroscópio. Coloque todos os instrumentos cirúrgicos afiados em uma bandeja resistente a perfurações para evitar lesões durante a cirurgia. </li><li> Esfregue todos os locais cirúrgicos 3 vezes com alternância de iodo e álcool 70% antes de realizar a incisão. Lave todos os cateteres com 30 solução de heparina salina UI / ml. </li><li> Faça de 1 - 1,5 centímetros incisão na pele na linha média sobre as escápulas usando um par de tesouras cirúrgicas sobre a veia jugular direita. Separa-se a fáscia por dissecção cega para expor a veia jugular e canular-lo com um tubo de polietileno (PE-50) cheio com heparina-solução salina utilizando 5-0 suturas de seda. Infundir anestesia suplementar quando necessário (1/3 a 1/2 da dose inicial, por via intravenosa (iv)) ao longo do curso da experiência e cirurgia. </li><li> Realização da traqueostomia para manter a desobstrução das vias aéreas. Adicione de 1 - 1,5 centímetros incisão na área cervical anterior. Canular a traqueia usando um tubo de polietileno (PE-205) com fio de seda 2-0 para fixar o cateter no local. </li><li> Monitor de pressão arterialatravés de punção na artéria femoral. Faça de 1 - 1,5 centímetros incisão na face medial esquerdo da perna traseira. Separar a artéria femoral por dissecção romba. Canular a artéria femoral, com um tubo de polietileno (PE-10) cheio com heparina-solução salina utilizando 5-0 suturas de seda. </li></ol></li><li> Cremaster Muscle Preparação and Flow Visualization <ol><li> Insira uma sutura 5-0 seda através do ápice do saco escrotal para estendê-lo. Realizar uma incisão ao longo da superfície ventral do saco escrotal. Regularmente aplicar a solução isotónica quente (37 ° C; pH 7,4) para o músculo exposta. </li><li> Remover circundante tecidos conjuntivos com cuidado e profundidade usando um aplicador com ponta de algodão. </li><li> Insira uma sutura 5-0 seda através do ápice do músculo cremaster. Corte a sutura em duas partes de igual comprimento e faça um nó em cada lado. Corte o músculo entre os dois nós e esticá-la em uma plataforma transparente de acrílico personalizado puxando suavemente a sutura. Consertara extremidade do fio de sutura para a plataforma com a aderência azul. NOTA: a remoção completa de cercar tecidos conjuntivos é crucial na obtenção de contraste de imagem ideal. </li><li> 1.2.3 Repita o passo até 5 a 6 fixações são feitas. Remova cuidadosamente o músculo cremaster do epidídimo usando alta cautério temperatura. Superfuse solução isotónica quente para o músculo exposta para evitar a desidratação do tecido. <ol><li> Cerque o músculo cremaster com peças dobradas de gaze. Cobrir o músculo exposta com uma película de polivinil. Os pedaços de gaze com o filme formar uma bacia rasa para segurar solução isotônica quente para o objetivo de imersão em água microscópio (Figura 1A). </li></ol></li><li> Transferir o animal para a fase de animais de um microscópio intravital (Figura 1C). Ligue a punção arterial a um sistema de aquisição de dados fisiológica para monitorização da pressão contínua (Figura 1E). </li><li> Manter a temperat muscularure a 35 ° C com um elemento de aquecimento ligado por baixo da plataforma do animal (Figura 1B). Coloque um sensor de temperatura junto ao músculo para fornecer feedback negativo para o controlador de energia do elemento de aquecimento (Figura 1D). </li><li> Deixar o animal no estágio durante 15 minutos para equilibrar com o ambiente. </li><li> Visualizar o fluxo sanguíneo ao microscópio intravital com um objetivo de imersão em água 40X e um condensador de funcionamento longa. </li><li> Escolha uma arteríola não ramificado (&lt;60 um), com base em um foco de imagem clara e contraste entre o núcleo de RBC, CFL e dos vasos paredes, a fim de focar o microscópio sobre o plano diametral do vaso sanguíneo. Rodar a câmara montada no microscópio para alinhar verticalmente a parede do vaso. </li><li> Grave o fluxo de sangue usando uma câmera de vídeo de alta velocidade com uma taxa de quadros de 3.000 / seg por 1 segundo. Salvar o vídeo gravado em formato AVI em tons de cinza não-comprimido de 8 bits para preservar a qualidade da imagem. NOTA:<html> Recomenda-se uma velocidade mínima do quadro de gravação de 3.000 quadros / seg para assegurar que a medição de CFL pode ser realizada pelo menos uma vez por RBC sob condições de fluxo fisiológicas arteriolar. </li><li> Usar um filtro azul com pico de transmissão em comprimento de onda de 394 nm, e passa-banda espectral de 310 - 510 nm, para aumentar o contraste entre as hemácias e plasma. NOTA: Certifique-se de que o espectro de luz que passa através do filtro azul da fonte de luz microscópica (lâmpada de halogéneo de 100 W) é de baixa intensidade de luz para evitar qualquer dano tecidual potencial. </li><li> No final da experiência, os animais a eutanásia com uma sobredose de pentobarbital de sódio. </li></ol></li></ol> Análise 2. Imagem <ol><li> Pré-processamento para a medição da largura do CFL <ol><li> Abra MATLAB e execute o arquivo &#39;CFL_pre.m&#39;. (Este e outros arquivos MATLAB pode ser encontrado noip &quot;&gt; Suplementar MATLAB Arquivo.) </li><li> Clique em &quot;Abrir arquivo&quot; para selecionar o arquivo de vídeo para analisar. </li><li> Ajuste o controle deslizante de &quot;rotação&quot; para alinhar as paredes dos vasos verticalmente. NOTA: Os usuários podem exibir as linhas de grade de apoio para o alinhamento navio, selecionando o botão de opção &#39;Grid On&#39; e ajuste o nível de zoom da imagem deslizando o controle deslizante &#39;Zoom&#39;. </li><li> Clique no botão &#39;Confirmar Editar&#39; para confirmar o alinhamento navio. </li><li> Clique no botão &#39;Definir ROI para a cultura&#39; para definir a região de interesse (ROI). A imagem alinhada será exibido em uma janela pop-up. Ajustar o objetivo rectangular na imagem e clique duas vezes para confirmar a ROI. Pule esta etapa se o corte de imagem não é necessária. NOTA: Apenas incluem um único recipiente na ROI para analisar a largura do recipiente de CFL. Clique no botão &quot;Reiniciar Imagem &#39;para restaurar a imagem à sua forma original, se necessário. </li><li> Clique no9; Extrato &#39;botão para extrair todos os quadros de vídeo editados em imagens de mapas de bits consecutivos (8-bit em tons de cinza &quot;Imagens BMP&#39;). As imagens extraídas podem ser encontrados na pasta com o mesmo nome do arquivo de vídeo selecionado. </li></ol></li><li> Medição da largura CFL <ol><li> Abra MATLAB e execute o arquivo &#39;CFL_measure.m&#39;. </li><li> Clique em &quot;Selecionar Pasta&quot; para selecionar a pasta que contém as imagens extraídas. </li><li> Clique na pasta que contém as imagens e clique em &quot;Selecionar Pasta&quot;. O primeiro quadro de imagem na pasta será carregado e mostrado no painel &#39;Tons de cinza imagem&#39;, juntamente com o seu cinza histograma de intensidade no painel &#39;Imagem Histograma&#39;. </li><li> Selecione o quadro de imagem desejada na caixa de lista para realizar a análise, caso contrário, o primeiro quadro de imagem será selecionado. </li><li> Clique &quot;Encontrar navio Paredes &#39;para identificar o interior da parede do vaso na imagem, que é determinada no local onde oluz perfil de intensidade de pico trânsitos de escuro para claro ao longo de dois pixels. </li><li> Verificar &#39;filtro mediano&#39; para aplicar um filtro de média para a imagem para reduzir o ruído de &quot;sal e pimenta&quot;. </li><li> Check &#39;Auto Contraste&#39; para ajustar as intensidades de imagem digital para aumentar o contraste da imagem. </li><li> Selecione um algoritmo de limiarização na caixa de listagem que determina automaticamente um valor limiar (τ) que divide os níveis de cinza em duas classes - pixels brancos com níveis de cinza acima τ (CFL), e pixels pretos com níveis de cinza abaixo τ (RBC core). NOTA: Como um método alternativo, use limiar manual se nenhum dos algoritmo de limiarização automatizado proporciona um limiar de imagem apropriado. Clique no botão de opção &#39;Manual&#39; e ajuste o controle deslizante para definir o valor de limiar manual. </li><li> Para medir a variação espacial das larguras CFL, digite a resolução de pixels na caixa de &#39;pixels de resolução&#39; (a resoluçãoCom esta configuração experimental foi de 0,42 mm / pixel). </li><li> Clique no botão &quot;Calcular&quot; para obter a variação espacial das larguras CFL. Clique em &#39;.csv Exportar&#39; para exportar os dados de largura CFL em um formato de tabela. </li><li> Para medir a variação temporal das larguras CFL em uma linha de análise específica ao longo do vaso, clique no botão de opção &#39;Temporal Variação &quot;e insira as informações taxa de quadros (a taxa de quadros usada nesta configuração experimental foi 3.000 frames / seg). </li><li> Digite o primeiro quadro e último quadro das imagens para a análise nas caixas &#39;Iniciar Frame&#39; e &#39;Última frame&#39;, respectivamente. </li><li> Seleccione a posição da linha de análise ao longo do vaso deslizando barra deslizante da &quot;Linha Análise &#39;. Confirmar a posição da linha de análise, o qual é ilustrado em ambos os &#39;imagem em escala de cinza &quot;e&quot; imagem binária&#39;. </li><li> Clique em &quot;Calcular&quot; para obter a variação temporal das larguras CFL. click &#39;Export .csv&#39; para exportar os dados de largura CFL em um formato de tabela. </li></ol></li></ol>

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The authors declare no competing financial interests.

A medição da largura CFL é essencial para uma melhor compreensão da hemodinâmica na microcirculação. Em particular, a medição de larguras CFL tem sido realizada em mesentérica 6, espinhotrapézio 24 e cerebrais 25 microcirculations. Medição convencional de in vivo larguras CFL foi restrita a estimativas por inspeção manual dos quadros de vídeo gravadas. As medições manuais necessários a média de vários quadros de vídeo sucessivas antes de identificar visualmente os limites das centrais e dos vasos paredes RBC 15,16. Em outro estudo, isotiocianato de fluoresceína (ITCF) marcado com RBCs e rodamina-B isotiocianato (RITC) de plasma marcadas foram utilizados para determinar as larguras médias CFL no CAT microvasos cerebrais 25. Estes métodos de medições anteriores são muito demorados e requerem passos adicionais para a marcação fluorescente, o que limita a resolução espacial e temporal do wi CFLmedição de DTH. Em contraste, por acoplamento de gravações câmara de alta velocidade a uma segmentação da imagem efectiva e a análise, a técnica demonstrada aqui permite a quantificação de variações temporais e espaciais da CFL com uma resolução espacial (0,42 mm) de uma ordem mais pequeno do que o tamanho de um RBC e uma resolução temporal de 1 / 3.000 seg. preparação cirúrgica adequada do músculo cremaster é crítica na determinação da precisão das medições de largura CFL. Em particular, a remoção completa dos tecidos conjuntivos adjacentes é essencial para garantir um bom foco das arteríolas do músculo cremaster. Além disso, a resolução espacial e temporal da medição depende das especificações do microscópio e a câmara. Embora um objectivo ampliação mais elevada pode melhorar a resolução espacial, que reduz o campo de visão, que por sua vez, limita o comprimento do vaso obtida para quantificar a variação espacial da largura de CFL. Portanto, o microscopiconfigurações C pode ser modificado de acordo com a aplicação específica da técnica. segmentação de imagens é outro fator importante para a precisão da medição da largura do CFL. De entre as várias técnicas desenvolvidas, limiar da imagem com base no nível de cinzento do histograma proporciona uma abordagem simples e eficaz para a segmentação e análise de imagem. Por conseguinte, objectos de fundo são extraídos do fundo com base na diferença entre os níveis de cinzento. No caso ideal, o histograma da imagem vai ser bimodal e um valor de limiar, na parte inferior do vale é trivial. No entanto, in vivo imagens experimentais nem sempre apresentam tais perfis de nível em tons de cinza. Nossos resultados têm mostrado como a qualidade da imagem e contraste podem influenciar o processo de segmentação de imagens. A utilização de um filtro azul óptica melhorada significativamente o contraste entre as hemácias e o plasma numa arteríola (Figura 3), e parece ser essencial quando applying o limiar com base em histograma para a medição da largura do CFL independentemente dos algoritmos (Figura 4). Isso resulta em um histograma da imagem bimodal distintos, o que permite identificar o valor do limiar de forma eficaz. No entanto, deve notar-se que, mesmo com um histograma bimodal obtidos a partir das imagens in vivo, uma variação extremamente desigual de dois picos (máximos locais) e um vale largo (mínimo local) do histograma ainda pode influenciar a selecção de limiar (Tabela 1 ). Portanto, a seleção de um algoritmo de limiar apropriada precisa ser examinado com base na qualidade da imagem e os usuários tem que considerar as limitações de cada algoritmo de limiarização para a melhor adequação na quantificação larguras CFL. Como as larguras CFL são largamente dependente das condições de escoamento, a medição da pressão arterial contínua ao longo do curso do experimento é essencial. A fim de determinar as condições de escoamento locais,a taxa pseudoshear do fluxo sanguíneo pode ser calculado através da medição da velocidade média de fluxo no vaso sanguíneo 5. Em resumo, os protocolos para a preparação cirúrgica de um músculo cremaster de rato e análise de imagem quantitativa descritos aqui foram utilizados para obter informação quantitativa sobre a variação dinâmica das larguras CFL in vivo. Os principais desafios para garantir a precisão dos CFL largura medições incluem preparação cirúrgica adequada da segmentação muscular e imagem, sendo que ambos foram abordados acima. Esta técnica pode ser facilmente adaptada para outros estudos para investigar a microcirculação e as aberrações hemorreológica hemodinâmicas em várias condições fisiológicas e patológicas. Consequentemente, estes resultados contribuem para o futuro desenvolvimento de abordagens terapêuticas microvasculares e intervenção clínica.

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

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			<td>.038&#34; OD X .023&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">PE-205 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427446</td>
			<td>.082&#34; OD X .062&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">2-0 non-absorbable silk suture</td>
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			<td height="21" style="height:21px;width:251px;">Sterile Cotton Gauze&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">22-415-468</td>
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			<td height="21" style="height:21px;width:251px;">Cotton-tipped applicators</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-124</td>
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			<td height="21" style="height:21px;width:251px;">Dumont Forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14188</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Micro Dissecting forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Iris forceps 1x2 teeth</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15917</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Vessel cannulation forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS500377</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Micro scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14177</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Iris scissor</td>
			<td style="width:167px;">Kent Scientific</td>
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			<td style="width:167px;">Kent Scientific</td>
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			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Gemini cautery system</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:119px;">GEM 5917</td>
			<td>Surgical instrument</td>
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visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

A visualização da CFL in vivo é largamente dependente das preparações cirúrgicos do animal. perda excessiva de sangue ou duração da cirurgia estendida pode sujeitar o animal ao choque e aberrações do fluxo sanguíneo. Manutenção da temperatura do tecido usando uma almofada de aquecimento, bem como uma plataforma de personalização durante a cirurgia e experiência também é crucial para manter as condições fisiológicas do rato. Por utilização de uma lâmpada de halogéneo de 100 W no sistema de microscópio, sem danos nos tecidos discernível foi observado mesmo no final da experiência. A Figura 2A mostra um típico fluxo GV através de uma arteríola não ramificado no músculo cremaster de rato, em que o CFL pode ser observado entre o núcleo de RBC e a parede do vaso interior (Figura 2C). Um bom contraste entre esses componentes durante a experiência é fundamental para garantir a precisão dos CFL medidas de largura. A fase inicial da análise de imagem envolve odetecção da parede do vaso interior. Ao adquirir o perfil intensidade de luz ao longo da linha de análise perpendicular ao navio, a localização é aproximada no pico que trânsitos de escuro para claro ao longo de dois pixels (Figura 2B). Como hemácias e CFL possuem diferentes transmissão da luz, a diferença de níveis de cinza pode ser subdividida em duas classes (imagem binária). No entanto, a identificação de um valor limite preciso entre os dois picos no histograma da imagem pode ser limitada por uma má qualidade de imagem e o contraste (Figura 3A). Para melhorar o contraste entre os GVs e CFL, um filtro azul pode ser utilizado (Figura 3B). Isto é também evidente na Figura 4, em que os limites do núcleo de glóbulos vermelhos pode ser identificado de forma mais precisa com a utilização de um filtro azul. Além disso, a selecção de algoritmo de limiar 20-23 também pode influenciar a medição da largura CFL (Figura4). É evidente na figura 4A que diferentes algoritmos de limiar resultou em diferentes fronteira núcleo RBC identificados, o que poderia levar a medições CFL erradas. Para melhor ilustrar a influência do algoritmo de limiar sobre a medição da largura do CFL na Figura 4B, os perfis espaciais para as larguras de CFL obtidos usando diferentes algoritmos de limiar são mostrados na Figura 5 e resumidos na Tabela 1. <img alt="figura 1" src="/files/ftp_upload/54550/54550fig1.jpg" /> Figura 1:. Intravital Sistema microscópica e Cremaster Preparação Muscle A:. Cirurgicamente exteriorizado músculo cremaster de rato B: Customized plataforma com elementos de aquecimento para colocar o músculo cremaster e manter a sua temperatura a 35 ° C C:. Sistema microscópico com cusmizadas palco animais e câmera de alta velocidade para a visualização dos fluxos de sangue da microcirculação no cremaster muscular D:. controlador de temperatura feedback negativo e fonte de alimentação E:. sistema de aquisição de dados fisiológicos para monitorização da pressão contínua. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54550/54550fig2.jpg" /> Figura 2: Processamento de Imagem para Determinação do navio parede Posição e CFL Largura A:.. Imagem em tons de cinza típico de fluxo RBC em uma arteríola (diâmetro do vaso = 52 mm) B: perfil de intensidade de luz ao longo da linha de análise (linha sólida no painel A) . C: resultado representativo de medida CFL ao longo do vaso. osetas a cheio e a tracejado indicam a parede do vaso interior e bordo exterior do núcleo de RBC, respectivamente. (LWB &amp; RWB: esquerda e direita navio fronteira parede, LCB &amp; RCB: esquerda e direita RBC núcleo de fronteira) <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54550/54550fig3.jpg" /> Figura 3:.. Aperfeiçoamento de contraste da imagem com um filtro azul Optical histograma da imagem das imagens em tons de cinza obtidos sem (A) e com filtro azul (B) <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54550/54550fig4.jpg" /> Figure 4: RBC Núcleo Largura Determinada utilizando cinco diferentes algoritmos de limiarização Fronteiras do núcleo RBC ea parede do reservatório sobreposto a imagens em tons de cinza na Figura 3.. (linha superior (A): sem filtro azul, linha de baixo (B): com filtro azul) usando (da esquerda para a direita) o método do Otsu, método mínimo, método intermode, método de seleção iterativa (Isodata) e limiar entrópica difusa (Shanbhag). As linhas contínuas e tracejadas indicam a parede do vaso interior e borda externa do núcleo RBC, respectivamente. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 5" src="/files/ftp_upload/54550/54550fig5.jpg" /> Figura 5: Variação espacial de CFL Largura CFL larguras correspondentes à Figura 4B ao longo da esquerda (A).e direito (B) paredes dos vasos, respectivamente. (D: distância em diâmetro do vaso) <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="tabela 1" src="/files/ftp_upload/54550/54550table1.jpg" /> Tabela 1: Valores limiar e CFL largura de dados na Figura 5. * p &lt;0,001: diferença significativa do método de Otsu. † p &lt;0,001: diferença significativa a partir da esquerda. As análises estatísticas foram realizadas utilizando testes t não pareado bicaudal.

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Visualização e quantificação da Camada de livre-Cell nas arteríolas do músculo Rat Cremaster

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

visualization-quantification-cell-free-layer-arterioles-rat-cremaster

Nanyang Technological University

Research

JoVE Journal

Biology

6.3K visualizações.  National University of Singapore. This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Vídeo: Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificação de γH2AX Focos em resposta à radiação ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Biologia

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificação de Progenitores do músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

O Mouse Preparação Muscle Cremaster for Imaging intravital da Microcirculação

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Imagem de receptor de estrogênio α-in Rat Arteríolas Pial usando um Microscópio Digital imunofluorescência

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital da Microcirculação no músculo Rato Cremaster de Análise de Migração Stem Peripheral celular

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Avaliação do cálcio Faíscas intactas fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

Quantificar Único microvasos Permeabilidade em perfusão isolada-Sangue Rat Lung Preparação

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Isolamento de Murino vasculares coronários células musculares lisas

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Um Protocolo Automatizado Rápido para Muscle Fiber Análise População em músculo secções transversais Usando Cadeia miosina pesada Imunohistoquímica

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...