We thank J. Hagoort for reading and correcting the manuscript.

Este protocolo segue as diretrizes do comitê de ética em pesquisa humanos local. 1. Preparação <ol><li> Marcar a medição da microcirculação de modo que ele não coincida com outros processos, tais como a amostragem de sangue. Em neonatos a termo que é melhor executada após a alimentação. Isto evita a agitação e vai facilitar a medição. </li><li> Certifique-se de que uma enfermeira ou um pai atende a apoiar e confortar o recém-nascido durante o exame, usando os princípios do Programa de Avaliação do recém-nascido 12 Individualizado Desenvolvimento e Cuidados. Nota: Embora as medições podem ser realizadas por uma única pessoa, é altamente recomendável para ter uma segunda pessoa auxiliar. Um prende a câmera e está focada no recém-nascido, enquanto o outro opera o computador e software. Em nossa experiência, isso resulta em imagens de alta qualidade e uma duração mais curta do procedimento. </li><li> Se a condição clínica dos neonatos licenças, coloque oneonato em posição supina. Imagem da microcirculação pode ser realizada em decúbito ventral, mas isso requer mais habilidade e paciência. </li><li> Verifique se a temperatura do corpo do recém-nascido prematuro está dentro do alcance adequado (36,5 - 37,5 graus Celsius). </li></ol> 2. Procedimento <ol><li> Instale o dispositivo ao longo da incubadora. Certifique-se da incubadora é a altura certa. </li><li> Coloque a tampa descartável na câmera. </li><li> Aplicar gel, óleo ou solução salina na ponta da sonda; isso vai ajudar a suavizar o contato entre a sonda ea pele. </li><li> Coloque a câmera no lado ventromedial do recém braço. Para evitar que o foco-artefactos, certifique-se a sonda é perpendicular à pele. Isto pode exigir o reposicionamento do braço lactentes. Nota: O lado ventromedial do braço é o local principal para medir a microcirculação cutânea. Este local tem pouco cabelo lanugo e é, portanto, menos propenso a artefatos. Ele é mais facilmente alcançadoSe o paciente é posicionado em posição supina. </li><li> Para minimizar o comprimento total do processo, ganhar tempo, encontrando a profundidade óptima de foco (Figura 3), enquanto procura o local com o menor número de produtos manufacturados. Nota: Profundidade do foco depende principalmente da idade pós-natal, em vez de idade gestacional. A profundidade média de foco na primeira semana de vida é de 0 - 80 um. Daqui por diante, devido ao amadurecimento da pele, a profundidade de foco aumenta rapidamente com valores médios de 80 - 200 um entre 1 - 4 semanas de idade pós-natal (Figura 2). No prazo nascido recém-nascidos a profundidade média de foco é 80-160 mm no nascimento. </li><li> Estabilizar a sonda para evitar artefatos de movimento. Para fazê-lo, o cotovelo descansar na janela da incubadora e o pulso ao lado do neonato. Alternativamente, posicionar a sonda ao lado do recém-nascido em um travesseiro. </li><li> Evite artefatos de pressão, permitindo que a câmara só tem a menor contato com a pele. Artefatos de pressão pode ser recognized durante captura de imagem se há fluxo de vai-e-vem em vasos ou se grandes vasos são não-perfundidos enquanto houver bom fluxo em pequenas embarcações. Além disso, se o padrão de fluxo é idêntica em toda a tela inteira, cuidado com os artefatos de pressão. </li><li> Gravar vídeos com uma duração mínima de 5 segundos. </li><li> Depois de uma captação bem sucedida, mova a câmera para outro local na parte superior do braço. Nota: Recomenda-se capturar um total de 5 - 10 vídeos no 3 - 5 locais diferentes, como alguns artefactos só são reconhecidos na análise off-line, o que significa que o vídeo em questão não é utilizável para análise. </li><li> Suavemente remover o gel, óleo ou solução salina a partir da pele com um pequeno gaze. </li></ol> 3. Análise offline <ol><li> Recortar o vídeo, se há um movimento significativo que impede a análise. Vá para a seção &#39;Ferramentas&#39; e use o botão &#39;Editor&#39;. Selecione o intervalo de quadro elegíveis para análise e clique no &#39;Vídeo da colheita&#39; button. Nota: Os vídeos são aceitáveis ​​se o movimento está dentro de ½ do campo de visão 13. </li><li> Selecione o vídeo recortada e estabilizá-lo. Vá para a seção &#39;Ferramentas&#39; e use o botão &#39;Análise&#39;. Clique no botão &quot;Estabilizar&quot;. Nota: Todos os filmes devem ser estabilizados antes da análise automática pode ser executada. </li><li> Selecione o vídeo estabilizado. Vá para a seção &quot;análise&quot; e clique no botão &#39;Detect&#39;. Certifique-se destacam as opções &#39;Capilares »e« navios ». </li><li> Após a detecção (Figura 4), ​​clique no botão &#39;De Backer&#39; &#39;CNA &quot;ou para um relatório completo da microcirculação. Este relatório inclui os parâmetros mais usados ​​de resultados como a densidade total do vaso (TVD), densidade de vasos perfundidos (PVD) e proporção de vasos perfundidos (PPV). Nota: Como alternativa, os vídeos podem ser exportados offline para ser analisada manualmente. Esta opção pode ser encontrada nas &#39;Ferramentas&#39; seção. Seleccione a opção &#39;Exportar&#39; e clique no botão &#39;AVA exportação ». </li></ol>

<ol>
	<li>Groner, W., 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=Orthogonal+polarization+spectral+imaging:+a+new+method+for+study+of+the+microcirculation.">Orthogonal polarization spectral imaging: a new method for study of the microcirculation.</a> Nat Med. 5 (10), 1209-1212 (1999).</li><li>Goedhart, P. T., Khalilzada, M., Bezemer, R., Merza, J., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sidestream+Dark+Field+(SDF)+imaging:+a+novel+stroboscopic+LED+ring-based+imaging+modality+for+clinical+assessment+of+the+microcirculation.">Sidestream Dark Field (SDF) imaging: a novel stroboscopic LED ring-based imaging modality for clinical assessment of the microcirculation.</a> Opt Express. 15 (23), 15101-15114 (2007).</li><li>Sherman, H., Klausner, S., Cook, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incident+dark-field+illumination:+a+new+method+for+microcirculatory+study.">Incident dark-field illumination: a new method for microcirculatory study.</a> Angiology. 22 (5), 295-303 (1971).</li><li>Trzeciak, S., 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=Early+microcirculatory+perfusion+derangements+in+patients+with+severe+sepsis+and+septic+shock:+relationship+to+hemodynamics,+oxygen+transport,+and+survival.">Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: relationship to hemodynamics, oxygen transport, and survival.</a> Ann Emerg Med. 49 (1), 88-98 (2007).</li><li>Sakr, Y., Dubois, M. J., De Backer, D., Creteur, J., Vincent, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+microcirculatory+alterations+are+associated+with+organ+failure+and+death+in+patients+with+septic+shock.">Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock.</a> Crit Care Med. 32 (9), 1825-1831 (2004).</li><li>De Backer, 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=Microcirculatory+alterations+in+patients+with+severe+sepsis:+impact+of+time+of+assessment+and+relationship+with+outcome.">Microcirculatory alterations in patients with severe sepsis: impact of time of assessment and relationship with outcome.</a> Crit Care Med. 41 (3), 791-799 (2013).</li><li>Buijs, E. 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=Early+microcirculatory+impairment+during+therapeutic+hypothermia+is+associated+with+poor+outcome+in+post-cardiac+arrest+children:+A+prospective+observational+cohort+study.">Early microcirculatory impairment during therapeutic hypothermia is associated with poor outcome in post-cardiac arrest children: A prospective observational cohort study.</a> Resuscitation. , (2013).</li><li>Genzel-Boroviczeny, O., Christ, F., Glas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+transfusion+increases+functional+capillary+density+in+the+skin+of+anemic+preterm+infants.">Blood transfusion increases functional capillary density in the skin of anemic preterm infants.</a> Pediatr Res. 56 (5), 751-755 (2004).</li><li>Ergenekon, E., 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=Peripheral+microcirculation+is+affected+during+therapeutic+hypothermia+in+newborns.">Peripheral microcirculation is affected during therapeutic hypothermia in newborns.</a> Arch Dis Child Fetal Neonatal Ed. 98 (2), F155-F157 (2013).</li><li>Schwepcke, A., Weber, F. D., Mormanova, Z., Cepissak, B., Genzel-Boroviczeny, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+mechanisms+in+postnatal+hypotension+affecting+premature+infants.">Microcirculatory mechanisms in postnatal hypotension affecting premature infants.</a> Pediatr Res. , (2013).</li><li>van Elteren, H. A., Ince, C., Tibboel, D., Reiss, I. K., de Jonge, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+microcirculation+in+preterm+neonates:+comparison+between+sidestream+dark+field+(SDF)+and+incident+dark+field+(IDF)+imaging.">Cutaneous microcirculation in preterm neonates: comparison between sidestream dark field (SDF) and incident dark field (IDF) imaging.</a> J Clin Monit Comput. , (2015).</li><li>Als, H., 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=Individualized+Behavioral+and+Environmental+Care+for+the+Very-Low-Birth-Weight+Preterm+Infant+at+High-Risk+for+Bronchopulmonary+Dysplasia+-+Neonatal+Intensive-Care+Unit+and+Developmental+Outcome.">Individualized Behavioral and Environmental Care for the Very-Low-Birth-Weight Preterm Infant at High-Risk for Bronchopulmonary Dysplasia - Neonatal Intensive-Care Unit and Developmental Outcome.</a> Pediatrics. 78 (6), 1123-1132 (1986).</li><li>Massey, M. J., 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=The+microcirculation+image+quality+score:+development+and+preliminary+evaluation+of+a+proposed+approach+to+grading+quality+of+image+acquisition+for+bedside+videomicroscopy.">The microcirculation image quality score: development and preliminary evaluation of a proposed approach to grading quality of image acquisition for bedside videomicroscopy.</a> J Crit Care. 28 (6), 913-917 (2013).</li><li>Weidlich, K., 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=Changes+in+microcirculation+as+early+markers+for+infection+in+preterm+infants--an+observational+prospective+study.">Changes in microcirculation as early markers for infection in preterm infants--an observational prospective study.</a> Pediatr Res. 66 (4), 461-465 (2009).</li><li>De Backer, 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=How+to+evaluate+the+microcirculation:+report+of+a+round+table+conference.">How to evaluate the microcirculation: report of a round table conference.</a> Crit Care. 11 (5), R101 (2007).</li><li>Sallisalmi, M., Oksala, N., Pettila, V., Tenhunen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+sublingual+microcirculatory+blood+flow+in+the+critically+ill.">Evaluation of sublingual microcirculatory blood flow in the critically ill.</a> Acta Anaesthesiol Scand. 56 (3), 298-306 (2012).</li><li>van den Berg, V. J., 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=Reproducibility+of+microvascular+vessel+density+analysis+in+Sidestream+dark-field-derived+images+of+healthy+term+newborns.">Reproducibility of microvascular vessel density analysis in Sidestream dark-field-derived images of healthy term newborns.</a> Microcirculation. 22 (1), 37-43 (2015).</li></ol>

Os autores não têm nada a revelar.

Nesse artigo, procuramos descrever e demonstrar a abordagem para geração de imagens da microcirculação transcutânea em recém-nascidos prematuros. Visualizando esse método irá ajudar os pesquisadores a dois dos maiores desafios na investigação: reprodutibilidade e o tempo eo trabalho natureza intensiva de aprender novas técnicas. Esta técnica pode proporcionar informação útil de microcirculação periférica em prematuros de forma não-invasiva. Medições em série pode ajudar os médicos a avaliar os efeitos de intervenções terapêuticas. A microcirculação é a área em que o passo final da cadeia de transporte de oxigénio ocorre. Estudos observacionais têm demonstrado uma relação entre a deterioração da microcirculação ao longo do tempo ea presença de infecções bacterianas 14. Como deterioração da microcirculação periférica é um dos primeiros sinais de sépsis em recém-nascidos prematuros, é geralmente colocada a hipótese de que a microcirculação periférica pode ajudar a prever o desenvolvimento desépsis. A qualidade das imagens depende principalmente as habilidades do operador. Embora os mais recentes dispositivos da câmara fornecer melhores especificações técnicas e de hardware, este ainda é de importância secundária. Os operadores devem ser devidamente treinado em imagem da microcirculação. Eles devem estar familiarizados com as recomendações da conferência de mesa-redonda realizada em Amsterdã em 2006 15 e com os critérios de qualidade desenvolvidos por Massey et al 13. Estes seis critérios (iluminação, duração, foco, o conteúdo, a estabilidade, a pressão) são a base de dados confiáveis ​​e de alta qualidade. Operadores inexperientes tendem a falhar em reconhecer freqüentemente pressão que ocorre artefactos 16. Note, porém, que estas normas e recomendações de qualidade foram elaborados para medição MI sublingual em adultos e não podem ser diretamente extrapolados para medições MI cutâneas em recém-nascidos prematuros. Os critérios de duração e conteúdo de qualidadesão, portanto, mais difícil de realizar. Além disso, é altamente recomendável para realizar as medições com dois operadores e pedir uma enfermeira ou dos pais para participar. A câmera deve ser mantida constante, artefatos de pressão deve ser evitado, o software deve ser operado e bem-estar da criança devem ser considerados. Especialmente se a criança se move muito, isso é demais para um operador. Por isso, é melhor ter uma pessoa lidar com a câmera e se concentrar na área de medição e outra pessoa operar o software e verificar os sinais vitais do bebê. Naturalmente, além dos aspectos técnicos do MI, a experiência na manipulação (críticos) recém-nascidos prematuros é essencial para os operadores. Apesar de seu caráter não-invasivo, MI ainda carrega um fardo e riscos em potencial. Limitar a duração das medições a um mínimo para evitar que a incubadora e, assim, a temperatura do corpo cai demasiado. Aplique sempre uma tampa de proteção descartável na câmeraantes das medições. A função desta tampa é dupla: Ele protege a pele contra o aquecimento potencial da ponta da sonda e serve como uma barreira artificial para prevenir a transmissão bacteriana. Além disso, equipamentos MI deve ser regularmente desinfectadas. Além disso, aplicam-se apenas uma pequena quantidade de gel ou óleo sobre a pele suavemente e removê-lo após a medição. Se mal utilizado, a combinação de petróleo e fototerapia terapêutica pode ter efeitos devastadores sobre a pele. Embora principalmente crianças dificilmente reagem com as medições, a instabilidade cardio-respiratória não pode ser excluída. Esta é a razão pela qual recomendamos a ter o bebê apoiado por um dos pais ou enfermeiro. Após a aquisição de dados, análise off-line dos vídeos deve ser realizada de um modo padronizado. Software comercial está disponível para analisar automaticamente vídeos MI acordo com as diretrizes 15. Análise manual é composto por três etapas. Primeiro, realce de contraste pode ser ap<html> dobraram a fim de atingir o contraste ideal. Em segundo lugar, vídeos devem ser estabilizadas. Esta etapa irá trazer para fora a importância da estabilidade durante as medições. Para a menor deriva pode fazer um vídeo inutilizável para análise. Em terceiro lugar, os navios devem ser tiradas e fluxo deve ser categorizada. Note que este passo final carrega um alto risco de má entre observador a variabilidade 17. Análise off-line manual deve ser realizada apenas por pesquisadores treinados. Existem dois métodos para relatar as medidas de resultados. O método convencional de análise da rede capilar (CNA) reflecte o comprimento total dos vasos dividido pela área de superfície medida (mm / mm 2). Alternativamente, a menos demorado De Backer Pontuação (DBS) pode ser usado. Nesse escore, três horizontal equidistante e três linhas verticais equidistantes são desenhados na tela. Densidade de vasos pode ser calculado como o número de navios que cruzam as linhas dividido pelo comprimento total das linhas (N / mm). _content &quot;&gt; Existem desvantagens para o campo da microcirculação. Heterogeneidade da microcirculação pesquisa faz é complicado estabelecer valores de referência. Por isso, a maioria das pesquisas são estudos observacionais ou comparações entre os grupos. A avaliação bastante complexo de vídeos da microcirculação e interferência humana subjetiva aumenta a possibilidade de uma fraca reprodutibilidade (Van den Berg, 2015 17). Por conseguinte, é importante que o método de obtenção de vídeos é padronizado. Para o futuro próximo, avanços técnicos irá melhorar a pesquisa em MI cutânea. Um exemplo é automatizado análise de computador das imagens de vídeo, que irá substituir a avaliação humana relativamente subjetiva de microvasos e assim excluem entre variabilidade observador. Um bom exemplo para padronizar esse campo de pesquisa é o uso de sensores de imagem sem fio, que medem a microcirculação em série no mesmo local exato. Isso fará com que MI cutâneamenos dependente do operador.

Hemodynamic diagnostics in critically ill preterm neonates has always been difficult. Most diagnostic tools used in adults cannot be applied in these tiny preterm infants; and then there is a problem of the sensitivity of the outcome parameters. But most of all, these infants are so vulnerable, that the risks of diagnostic procedures do not outweigh the benefits. As a result, in the field of neonatology, hemodynamics has been neglected and therefore there is a lack of knowledge on this topic.
An interesting option for handling these problems might be visualizing the microcirculation. The introduction of handheld microscopes in the late 1990s made it possible to visualize the microcirculation in a non-invasive manner. Three generations of devices have been introduced: Orthogonal Polarization Spectral (OPS) imaging1, Sidestream Dark Field (SDF) imaging2, and Incident Dark Field (IDF) imaging3. They all use more or less the same technique in which green light with a specific wavelength (548nm) stroboscopic illuminates the microcirculation. The green light is absorbed by oxy- and deoxyhemoglobin and mostly reflected by the surrounding tissue. This property of green light therefore creates visible contrast. The reflected light passes a magnification lens and is projected on a camera sensor. Hereby it is possible to visualize the flowing red blood cells at a depth of approximately one millimeter of mucosal tissue or directly at solid organs. 
Over the past 15 years, the microcirculation has been mainly studied in adults, especially in patients with septic shock4-6. These observational studies found that persistent microcirculatory alterations were associated with organ failure and mortality. This observation cannot be extrapolated directly to (preterm) infants however, as in the adults the microcirculation was measured sublingually. High quality images of the sublingual microcirculation cannot be obtained in preterm infants because they are unable to cooperate. In term infants the buccal microcirculation has been the area of interest7. Fortunately, in preterm infants the thin skin allows transcutaneous microcirculatory imaging. This approach has been applied in neonatal studies focusing on blood transfusion8, therapeutic hypothermia9 and hypotension10.
In this manuscript we present our protocol for transcutaneous microcirculatory imaging using Incident Dark Field imaging in preterm neonates. We will focus on different strategies to acquire the highest quality images. Technical details and differences between the SDF and IDF devices can be found elsewhere11.

<table><tbody><tr><td>Cytocam</td><td>Braedius</td><td>http://www.braedius.com/magnoliaPublic/braedius/products.html</td><td>Other well known handheld microscopes to visualize the microcirculation are MicroScan (Microvision Medical) using SDF technique or the CytoScan (CytoMetrics) using OPS technique</td></tr><tr><td>Disposable Lens Cover</td><td>Glycocheck</td><td>http://www.glycocheck.com/lenscovers.php</td><td></td></tr><tr><td>CCTools</td><td>Braedius</td><td>http://www.braedius.com/magnoliaPublic/braedius/products.html</td><td>Another well known offline analysis programme is AVA (Microvision medical).&amp;nbsp;</td></tr></tbody></table>

transcutaneous microcirculatory imaging in preterm neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

As Figuras 1 e 2 mostram representante de imagens fixas de alta qualidade vídeos mi. Estes exemplos demonstram a diferença da espessura da pele em crianças entre os mesmos dia 1 (Figura 1) e no dia 28 (Figura 2) da idade pós-natal. No dia 1, há uma iluminação brilhante, foco adequada sobre os micro vasos e presença mínima de artefatos. No dia 28, é mais difícil encontrar o equilíbrio certo entre foco nas micro vasos e artefatos devido à pele mais grossa. Note-se que a estabilidade, duração e artefatos de pressão não pode ser julgado sobre essas imagens estáticas. Isso deve ser observado durante a aquisição das imagens ou antes da análise offline. <img alt="figura 1" src="/files/ftp_upload/53562/53562fig1.jpg" /> Figura 1. MI de prematuros 24 semanas Idade Gestacional, Dia 1. transcutânea MI de um recém-nascido com uma idade gestacional de 24 semanas. A idade pós-natal do recém-nascido é de 1 dia. A profundidade de foco usada é de 40 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53562/53562fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53562/53562fig2.jpg" /> Figura 2. MI de prematuros 24 semanas de idade gestacional, dia 28. transcutânea MI de um recém-nascido com uma idade gestacional de 24 semanas. A idade pós-natal do recém-nascido é de 28 dias. A profundidade de foco usada é de 160 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53562/53562fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53562/53562fig3.jpg" /> Figura 3. Diferenças é Foco Depth. Estas duas imagens de exatamente a mesma área de destacar a importância da profundidade foco adequado. Foco (esquerda) resultados inadequados em perda de visibilidade navio em comparação com o foco adequado (à direita). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53562/53562fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53562/53562fig4.jpg" /> Figura 4. Resultados da análise automática off-line. Esta figura mostra os resultados da análise off-line automática. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53562/53562fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Transcutaneous Microcirculatory Imaging in Preterm Neonates at JoVE.com

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Transcutânea Microcirculatory Imaging no recém-nascidos pré-termo

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

transcutaneous-microcirculatory-imaging-in-preterm-neonates

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.1K visualizações.  Erasmus MC-Sophia Children's Hospital. Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Vídeo: Transcutaneous Microcirculatory Imaging in Preterm Neonates

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Não-invasivo de medição óptica do metabolismo cerebral e Hemodinâmica em Lactentes

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Medicina

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

Avaliação não invasiva da função microvascular e endotelial

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

Um método para fazer uma craniotomia no crânio ventral de neonatos Roedores

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Neurociência

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

Estado da Arte Cranial Ecografia em recém-nascidos

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Análise quantitativa de Micro-CT de Aortopathy em um modelo do rato de β-aminopropionitrile-induzida de aneurisma da aorta e dissecação

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact morbidity and mortality rates. However, there is currently a lack of suitable non-invasive, real-time technologies for continuous monitoring of cerebral physiology at the bedside. Diffuse optical techniques have been proposed as a potential tool for bedside measurements of cerebral blood flow and cerebral oxygenation in case of neurocritical patients. Diffuse optical spectroscopies have been previously explored to monitor patients in several clinical scenarios ranging from neonatal monitoring to cerebrovascular interventions in adults. However, the feasibility of the technique to aid clinicians by providing real-time information at the bedside remains largely unaddressed. Here, we report the translation of a diffuse optical system for continuous real-time monitoring of cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism during intensive care. The real-time feature of the instrument could enable treatment strategies based on patient-specific cerebral physiology rather than relying on surrogate metrics, such as arterial blood pressure. By providing real-time information on the cerebral circulation at different time scales with relatively cheap and portable instrumentation, this approach may be especially useful in low-budget hospitals, in remote areas and for&#160;monitoring in open fields (e.g., defense and sports).

Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism.

Monitoramento em Tempo Real de Pacientes Neurocríticos com Espectroscopias Ópticas Difusas

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Avaliação em tempo real da microperfusão da medula espinhal em um modelo suíno de isquemia/reperfusão

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Hemodynamic Precision in the Neonatal Intensive Care Unit using Targeted Neonatal Echocardiography

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, reliable, and real-time information on developmental hemodynamics in sick newborns. The comprehensive assessment is based on a multiparametric approach that overcomes the reliability issues of individual measurements, allows for earlier recognition of cardiovascular compromise and promotes enhanced diagnostic precision and timely management. TnECHO-driven research has led to an enhanced understanding of the mechanisms of illness and the development of predictive models to identify at-risk populations. This information may then be used to formulate a diagnostic impression and provide individualized guidance for the selection of cardiovascular therapies. TnECHO is based on the expert consultative model in which a neonatologist, with advanced training in neonatal hemodynamics, performs comprehensive and standardized TnECHO assessments. The distinction from point of care ultrasonography (POCUS), which provides limited and brief one-time assessments, is important. Neonatal hemodynamics training is a 1-year structured program designed to optimize image acquisition, measurement analysis, and hemodynamic knowledge (physiology, pharmacotherapy) to support cardiovascular decision-making. Neonatologists with hemodynamic expertise are trained to recognize deviations from normal anatomy and appropriately refer cases of possible structural abnormalities. We provide an outline of neonatal hemodynamics training, the standardized TnECHO imaging protocol, and an example of representative echo findings in a hemodynamically significant patent ductus arteriosus.

Presented here is a protocol to perform comprehensive neonatal echocardiography by trained neonatologists in the neonatal intensive care unit. The trained individuals provide longitudinal assessments of heart function, systemic and pulmonary hemodynamics in a consultative role. The manuscript also describes the requirements to become a fully trained neonatal hemodynamics specialist.

Precisão Hemodinâmica na Unidade de Terapia Intensiva Neonatal utilizando Ecocardiografia Neonatal Direcionada

Targeted neonatal echocardiography (TnECHO) refers to the use of comprehensive echocardiographic evaluation and physiologic data to obtain accurate, ...

Non-Invasive Monitoring of Microvascular Oxygenation and Reactive Hyperemia using Hybrid, Near-Infrared Diffuse Optical Spectroscopy for Critical Care

The detection of levels of impairment in microvascular oxygen consumption and reactive hyperemia is vital in critical care. However, there are no practical means for a robust and quantitative evaluation. This paper describes a protocol to evaluate these impairments using a hybrid near-infrared diffuse optical device. The device contains modules for near-infrared time-resolved and diffuse correlation spectroscopies and pulse-oximetry. These modules allow the non-invasive, continuous, and real-time measurement of the absolute, microvascular blood/tissue oxygen saturation (StO2) and the blood flow index (BFI) along with the peripheral arterial oxygen saturation (SpO2). This device uses an integrated, computer-controlled tourniquet system to execute a standardized protocol with optical data acquisition from the brachioradialis muscle. The standardized vascular occlusion test (VOT) takes care of the variations in the occlusion duration and pressure reported in the literature, while the automation minimizes inter-operator differences. The protocol we describe focuses on a 3-min occlusion period but the details described in this paper can readily be adapted to other durations and cuff pressures, as well as other muscles. The inclusion of an extended baseline and post-occlusion recovery period measurement allows the quantification of the baseline values for all the parameters and the blood/tissue deoxygenation rate that corresponds to the metabolic rate of oxygen consumption. Once the cuff is released, we characterize the tissue reoxygenation rate, magnitude, and duration of the hyperemic response in BFI and StO2. These latter parameters correspond to the quantification of the reactive hyperemia, which provides information about the endothelial function. Furthermore, the above-mentioned measurements of the absolute concentration of oxygenated and deoxygenated hemoglobin, BFI, the derived metabolic rate of oxygen consumption, StO2, and SpO2 provide a yet-to-be-explored rich data set that can exhibit disease severity, personalized therapeutics, and management interventions.

We describe a protocol to non-invasively and continuously measure absolute microvascular blood flow index and blood oxygen saturation using a multi-modal device based on near-infrared diffuse optics. We then evaluate the metabolic rate of oxygen consumption and reactive hyperemia utilizing a vascular occlusion test.

Monitoramento Não Invasivo da Oxigenação Microvascular e Hiperemia Reativa por Espectroscopia Óptica Difusa Híbrida no Infravermelho Próximo para Terapia Intensiva