Projeto nº. NVKP_16-1-2016-0017 ('Programa Nacional do Coração') foi implementado com o apoio do Fundo Nacional de Pesquisa, Desenvolvimento e Inovação da Hungria, financiado sob o regime de financiamento NVKP_16. A pesquisa foi financiada pelo Programa de Excelência Temática (2020-4.1.1.-TKP2020) do Ministério da Inovação e Tecnologia na Hungria, no âmbito dos programas temáticos de Desenvolvimento Terapêutico e Bioimagem da Universidade Semmelweis.

Este protocolo segue as diretrizes do Comitê Regional e Institucional de Ética em Ciência e Pesquisa da Universidade Semmelweis. O presente protocolo se aplica a um fornecedor específico. Embora algumas etapas permaneçam válidas independentemente da máquina de ultrassom e do software de pós-processamento, podem existir diferenças importantes se usar as soluções de outros fornecedores.
1. Requisitos técnicos
<ol><li>Utilize uma máquina de ecocardiografia capaz de imagens 3D.</li>
	<li>Conecte um transdutor de matriz transtorácica 3D capaz de fases.</li>
	<li>Aplique o ECG embutido de 3 chumbo do sistema de ultrassom para permitir que o sistema sincronize as gravações e análises ao ciclo cardíaco.</li>
</ol>2. Aquisição das imagens ecocardiográficas 3D
<ol><li>Posicione o paciente na posição de decúbito lateral esquerdo (paciente deitado no lado esquerdo com o braço esquerdo esticado acima da cabeça).</li>
	<li>Certifique-se de que o rastreamento do ECG na tela é de boa qualidade. NOTA: Este é um pré-requisito para o pós-processamento, pois o software detectará os diferentes pontos do ciclo cardíaco com base no sinal ECG.</li>
	<li>Descongele a imagem e comece a examinar o paciente com o transdutor. Visualize uma visão apical convencional de quatro câmaras.</li>
	<li>Otimize a qualidade da imagem ajustando a largura do setor para LV, diminuindo a profundidade para truncar o átrio esquerdo e usando um ligeiro sobregain. NOTA: Certifique-se de que toda a LV e também a superfície epicárardial são visíveis.</li>
	<li>Pressione o botão 4D para mudar para o modo 3D. NOTA: Ao pressionar o botão Multi-Slice... na tela sensível ao toque, quatro opções estarão disponíveis (5, 7, 8, 12 fatias) para visualizar demais o conjunto de dados 3D usando cortes de eixo curto e longo padrão. Se necessário, o posicionamento do transdutor pode ser corrigido para garantir a inclusão de toda a espessura da parede LV do nível apical a mitral da válvula no conjunto de dados 3D piramim. Recomenda-se o uso de 12 fatias (com nove visualizações ajustáveis de eixo curto).</li>
	<li>Adquira imagens 3D usando o modo Multi Beat ou Single Beat .<ol>
<li>Use o modo Multi Beat para obter maior resolução espacial e temporal, onde o conjunto de dados será reconstruído a partir de 2, 3, 4 ou 6 ciclos cardíacos (isso pode ser configurado na tela) - respiração expiratória final do paciente e posicionamento transdutor estável necessário para minimizar artefatos de costura. NOTA: A aquisição do Single Beat é de menor resolução espacial e temporal; no entanto, a maioria dos transdutores modernos têm melhor qualidade e, portanto, podem ser usados para adquirir conjuntos de dados 3D adequados sem reconstrução para passar por análises mais aprofundadas. Como recomendação geral, são recomendadas taxas de volume acima de 15 volumes por segundo para análise suplementar.</li>
		<li>Quando o volume total for reconstruído a partir dos subvolumes, e toda a LV estiver visível, congele a imagem. Usando os botões Cycle Select e Number of Cycles , selecione os ciclos cardíacos adquiridos ideais e pressione a Image Store. NOTA: Os artefatos de costura são subvolumes espacial ou temporariamente desalinhados próximos um do outro. Conjuntos de dados com uma queda significativa de paredes LV ou com artefatos de costura geralmente não são adequados para análises adicionais. A qualidade do conjunto de dados 3D já adquirido pode ser verificada duas vezes usando o modo Multi-Slice.</li>
	</ol>
</li>
</ol>3. Pós-processamento para quantificar a morfologia e a função de LV
<ol><li>Selecione um conjunto de dados 3D apropriado para análise suplementar. NOTA: Esta parte do protocolo requer as imagens 3D adquiridas e salvas previamente adquiridas e salvas de boa qualidade e pode ser realizada na máquina de ultrassom e em uma estação de trabalho separada, também.</li>
	<li>Clique em Measure | Volume e, em seguida, selecione 4D Auto LVQ.</li>
	<li>Na tela quádrupla (três visões apical: vistas de quatro, duas e três câmaras e uma visão de eixo curto, esta última pode ser ajustada por um plano horizontal em vistas de longo eixo), o software pede modificar o alinhamento de fatias apical às visualizações padrão. Se necessário, corrija manualmente as visualizações a apical inclinando e rotação para mostrar a visão padrão correspondente, eliminando assim o estreitamento. Coloque inclinando para alinhar a pinça com o eixo longo da LV arrastando e movendo as pinças em vistas de longo eixo. Defina a rotação pelo correspondente ou pelo Girador Todos os botões da máquina ou ajustando as pinças na imagem do eixo curto. NOTA: A recomendação do software pode ser redefinida pressionando o botão Auto Align .</li>
	<li>Após o término do alinhamento de exibição, clique na próxima etapa EDV. O quadro end-diastólico (ED) é detectado automaticamente usando o sinal ECG, mas pode ser corrigido manualmente, se necessário.</li>
	<li>Detecção semiautomática de superfície endo e epicárida de LV<ol><li>Selecione dois pontos de referência manualmente em qualquer exibição apical. Em primeiro lugar, identifique o ápice lv e, em seguida, o meio da base LV (nível de anulo mitral) em qualquer visão apical. O algoritmo contornará automaticamente a borda endocárrica de toda a LV. NOTA: Há mais duas opções: Manual, o que significa que dois marcos basais e um apical devem ser definidos em todas as exibições apical, e Auto Init, que irá contornar automaticamente o LV sem qualquer interação do usuário.</li>
		<li>Verifique a credibilidade do contorno em três visões apical, três visões de eixo curto de diferentes níveis e um quarto eixo curto controlado pelo usuário, para permitir a verificação visual da superfície detectada. A correção do contorno é possível adicionando manualmente pontos que serão incorporados na linha de contorno. NOTA: Com o Desfazer, o ponto adicionado anteriormente pode ser excluído. O botão de reset redefine o contorno para iniciar toda a seção desde o início. A visibilidade do contorno pode ser ajustada para permitir a apreciação da superfície do endocárdio na imagem em escala cinza. O contorno endocárdio e epicoardial deve ser realizado de forma precisa e consistente. Para obter uma recomendação detalhada, verifique a seguinte referência6.</li>
		<li>Escolha o próximo passo, que é o ESV.</li>
		<li>Repita o mesmo procedimento (3.5.1-3.5.2) como mencionado nos pontos anteriores para identificar e corrigir o contorno endocárdial no quadro end-sistólico. NOTA: O quadro end-sistólico (ES) é detectado automaticamente usando o sinal ECG, mas pode ser corrigido manualmente, se necessário. Os valores do volume de dia diastólica final (EDV), volume sistólico final (ESV), fração de ejeção (EF), frequência cardíaca (RH), volume de derrame (SV), saída cardíaca (CO) e índice de esferografia (IPE) já são exibidos na tela.</li>
		<li>Pressione a forma de onda volume para o próximo passo. O software exibe um modelo 3D dinâmico do LV e também a curva de volume de tempo à medida que traça a superfície do endocárdio ao longo do ciclo cardíaco quadro por quadro (Figura 1). NOTA: Aqui, há a possibilidade de editar a borda do endocárdio em qualquer quadro.</li>
		<li>Para o próximo passo, pressione LV Mass. O software contorna automaticamente o contorno epictárido LV no quadro de diastólica final e calcula a massa LV (EDMass). NOTA: Se necessário, edite o contorno da superfície epicárardia adicionando pontos a incluir (mesmo método descrito anteriormente) em qualquer plano de curto ou longo eixo. Pode ser selecionado qual contorno ajustar: Endo, Epi ou Endo+Epi.</li>
		<li>Pressione o ROI de tensão 4D para o próximo passo. O software contorna automaticamente o contorno epictárário LV no quadro sistólica final e calcula a massa sistólica final da LV (ESMass). NOTA: Se necessário, edite o contorno sistólico final da superfície epicárardial adicionando pontos a incluir (mesmo método descrito anteriormente) em qualquer plano de eixo curto ou longo. O ESMass deve ser de valor semelhante ao EDMass. Esta etapa é essencial para calcular valores de tensão 3D por rastreamento de manchas.</li>
		<li>Pressione os resultados da tensão 4D para a próxima etapa. O software visualiza o rastreamento do miocárdio 3D em vários planos de curto e longo eixo e valores correspondentes de tensão dos 17 segmentos de LV padrão ao longo do ciclo cardíaco, quadro por quadro. Curvas de tensão temporal e gráfico de olho de touro também são exibidas. Os seguintes parâmetros são calculados e podem ser demonstrados: tensão longitudinal, tensão circunferencial, tensão radial, tensão da área, rotação e torção. NOTA: Existe a possibilidade de excluir um determinado segmento de LV da análise se for considerado como tendo baixa qualidade de rastreamento por observação visual de imagens ou com base na curva de tensão temporal. No entanto, o software recomenda por padrão a aprovação ou rejeição do segmento. Os valores de tensão codificados por cores podem ser visualizados em um modelo 3D dinâmico da LV alterando o "Layout".</li>
	</ol></li>
	<li>Para encerrar a análise, pressione Approve & Exit.</li>
</ol>

<ol>
	<li>Guta, A. C., 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=Three-dimensional+echocardiography+to+assess+left+ventricular+geometry+and+function.">Three-dimensional echocardiography to assess left ventricular geometry and function.</a> Expert Review of Cardiovascular Therapy. 17 (11), 801-815 (2019).</li><li>Surkova, 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=Current+Clinical+Applications+of+Three-Dimensional+Echocardiography:+When+the+Technique+Makes+the+Difference.">Current Clinical Applications of Three-Dimensional Echocardiography: When the Technique Makes the Difference.</a> Current Cardiology Reports. 18 (11), 109 (2016).</li><li>Lang, R. M., 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=Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Journal of the American Society of Echocardiography. 28 (1), 1-39 (2015).</li><li>Matyas, C., 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=Comparison+of+speckle-tracking+echocardiography+with+invasive+hemodynamics+for+the+detection+of+characteristic+cardiac+dysfunction+in+type-1+and+type-2+diabetic+rat+models.">Comparison of speckle-tracking echocardiography with invasive hemodynamics for the detection of characteristic cardiac dysfunction in type-1 and type-2 diabetic rat models.</a> Cardiovascular Diabetology. 17 (1), 13 (2018).</li><li>Kovacs, 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=Impact+of+hemodialysis,+left+ventricular+mass+and+FGF-23+on+myocardial+mechanics+in+end-stage+renal+disease:+a+three-dimensional+speckle+tracking+study.">Impact of hemodialysis, left ventricular mass and FGF-23 on myocardial mechanics in end-stage renal disease: a three-dimensional speckle tracking study.</a> International Journal of Cardiovascular Imaging. 30 (7), 1331-1337 (2014).</li><li>Muraru, 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=Comprehensive+analysis+of+left+ventricular+geometry+and+function+by+three-dimensional+echocardiography+in+healthy+adults.">Comprehensive analysis of left ventricular geometry and function by three-dimensional echocardiography in healthy adults.</a> Journal of the American Society of Echocardiography. 26 (6), 618-628 (2013).</li><li>Lakatos, B. 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=Relationship+between+Cardiac+Remodeling+and+Exercise+Capacity+in+Elite+Athletes:+Incremental+Value+of+Left+Atrial+Morphology+and+Function+Assessed+by+Three-Dimensional+Echocardiography.">Relationship between Cardiac Remodeling and Exercise Capacity in Elite Athletes: Incremental Value of Left Atrial Morphology and Function Assessed by Three-Dimensional Echocardiography.</a> Journal of the American Society of Echocardiography. 33 (1), 101-109 (2020).</li><li>Muraru, 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=Intervendor+Consistency+and+Accuracy+of+Left+Ventricular+Volume+Measurements+Using+Three-Dimensional+Echocardiography.">Intervendor Consistency and Accuracy of Left Ventricular Volume Measurements Using Three-Dimensional Echocardiography.</a> Journal of the American Society of Echocardiography. 31 (2), 158-168 (2018).</li><li>Kalam, K., Otahal, P., Marwick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+implications+of+global+LV+dysfunction:+a+systematic+review+and+meta-analysis+of+global+longitudinal+strain+and+ejection+fraction.">Prognostic implications of global LV dysfunction: a systematic review and meta-analysis of global longitudinal strain and ejection fraction.</a> Heart. 100 (21), 1673-1680 (2014).</li><li>Muraru, 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=Validation+of+a+novel+automated+border-detection+algorithm+for+rapid+and+accurate+quantitation+of+left+ventricular+volumes+based+on+three-dimensional+echocardiography.">Validation of a novel automated border-detection algorithm for rapid and accurate quantitation of left ventricular volumes based on three-dimensional echocardiography.</a> European Journal of Echocardiography. 11 (4), 359-368 (2010).</li><li>Doronina, 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=The+Female+Athlete's+Heart:+Comparison+of+Cardiac+Changes+Induced+by+Different+Types+of+Exercise+Training+Using+3D+Echocardiography.">The Female Athlete's Heart: Comparison of Cardiac Changes Induced by Different Types of Exercise Training Using 3D Echocardiography.</a> BioMed Research International. 2018, 3561962 (2018).</li><li>Takeuchi, M., 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=Measurement+of+left+ventricular+mass+by+real-time+three-dimensional+echocardiography:+validation+against+magnetic+resonance+and+comparison+with+two-dimensional+and+m-mode+measurements.">Measurement of left ventricular mass by real-time three-dimensional echocardiography: validation against magnetic resonance and comparison with two-dimensional and m-mode measurements.</a> Journal of the American Society of Echocardiography. 21 (9), 1001-1005 (2008).</li><li>Armstrong, A. 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As medições morfológicas e funcionais representam pilares do diagnóstico, do manejo e do acompanhamento de doenças cardíacas; além disso, eles são poderosos preditores de resultado. Geralmente, a avaliação baseada em ecocardiografia 2D do LV é recomendada pelas diretrizes atuais da prática; no entanto, a ecocardiografia 3D tem se mostrado mais precisa, pois está livre de suposições geométricas relativas à forma lv7,8. A deformação por rastreame...

A avaliação da morfologia e função ventricular esquerda (LV) é o propósito predominante de investigações gerais e ainda mais específicas em cardiologia1. A ecocardiografia transtorácica amplamente disponível e não invasiva (TTE), que pode fornecer quantidades densas de informações, é o método de escolha para uma avaliação conveniente, rápida e econômica.
A medição da massa lv, volumes e fração de ejeção subsequente contém significativo valor diagnóstico e também prognóstico2. Quanto mais precisa for uma determinada medida, maior será seu valor. Uma melhor correlação com os valores derivados da ressonância magnética cardíaca padrão-ouro (RMC) é uma perseguição contínua por técnicas ecocardiográficas. Geralmente, as diretrizes de prática clínica recomendam o método do biplano Simpson para medição de volume de LV e ejeção de frações3. No entanto, o LV é uma estrutura tridimensional (3D) com uma forma muitas vezes irregular, e, portanto, vários planos tomográficos, sem dúvida, falharão em alguns cenários clínicos para delinear com precisão a morfologia e a função da LV. Os recentes avanços na tecnologia ultrassônica de hardware e software permitiram o desenvolvimento de imagens 3D em tempo real, o que revoluciona os protocolos ecocardiográficos.
Além disso, a necessidade de uma abordagem quantitativa sobre anormalidades no movimento da parede resultou no aumento da imagem de deformação4. Os parâmetros de tensão e taxa de tensão podem ser calculados pelo rastreamento de manchas usando imagens padrão em escala cinza. A ecocardiografia 3D também pode superar várias deficiências de uma avaliação de tensão bidimensional5. A partir de uma ferramenta científica cara, a ecocardiografia 3D começou a se tornar uma técnica poderosa usada na prática clínica cotidiana, e a quantificação da LV está certamente na primeira linha neste avanço.
O presente artigo tem como objetivo orientar o leitor através de um protocolo 3D detalhado, apresentando dicas e truques e também destacando as potenciais armadilhas para facilitar o uso generalizado, mas tecnicamente sólido desta importante técnica em relação à LV.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3V-D/4V-D/4Vc-D</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>ultrasound probe</td>
		</tr>
		<tr>
			<td>4D Auto LVQ</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>software for analysis</td>
		</tr>
		<tr>
			<td>E9/E95</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>ultrasound machine</td>
		</tr>
		<tr>
			<td>EchoPac v203</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>software for analysis</td>
		</tr>
	</tbody>
</table>

evaluation of left ventricular structure and function using 3d echocardiography

A quantificação tridimensional (3D) do ventrículo esquerdo (LV) fornece valor agregado significativo em termos de precisão diagnóstica e estratificação precisa de risco em vários distúrbios cardíacos. Recentemente, a ecocardiografia 3D tornou-se disponível na prática de cardiologia de rotina; no entanto, a aquisição de imagens de alta qualidade e a análise subsequente têm uma curva de aprendizado íngreme. O presente artigo tem como objetivo orientar o leitor através de um protocolo 3D detalhado, apresentando dicas e truques e também destacando as potenciais armadilhas para facilitar o uso generalizado, mas tecnicamente sólido desta importante técnica em relação à LV. Em primeiro lugar, mostramos a aquisição de um conjunto de dados 3D de alta qualidade com resolução espacial e temporal ideal. Em seguida, apresentamos os passos analíticos para uma quantificação detalhada da LV usando um dos softwares embutidos mais amplamente aplicados. Quantificaremos os volumes de LV, a esficidade, a massa e também a função sistólica medindo a fração de ejeção e a deformação do miocárdio (cepa longitudinal e circunferencial). Discutiremos e forneceremos exemplos clínicos sobre os cenários essenciais onde a transição de uma abordagem ecocardiográfica convencional para uma quantificação baseada em 3D é altamente recomendada.

A análise 3D da LV é viável na maioria dos pacientes. O caso 1 é um voluntário saudável com volumes e função ventriculares normais (Figura 1). O caso 2 (Figura 2) é um paciente do sexo masculino de 64 anos com cardiomiopatia dilatada e um amplo complexo QRS (160 ms) de morfologia do bloco do ramo do feixe esquerdo. As medidas de CMR padrão ouro foram as seguintes: volume de dianólica final: 243 mL, volume sistólico fi...

Watch this Scientific Journal Video about Evaluation of Left Ventricular Structure and Function using 3D Echocardiography at JoVE.com

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Neste artigo, fornecemos um protocolo passo-a-passo de aquisição e análise para a avaliação volumostrica e análise de rastreamento de manchas do ventrículo esquerdo por ecocardiografia 3D, particularmente com foco em aspectos práticos que maximizam a viabilidade dessa técnica.

Avaliação da Estrutura e Função Ventricular Esquerda utilizando Ecocardiografia 3D

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk ...

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Universidad de Cartagena UDEC

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Bioengineering

3.8K visualizações.  Semmelweis University. Neste artigo, fornecemos um protocolo passo-a-passo de aquisição e análise para a avaliação volumostrica e análise de rastreamento de manchas do ventrículo esquerdo por ecocardiografia 3D, particularmente com foco em aspectos práticos que maximizam a viabilidade dessa técnica.

Vídeo: Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Pressão Ventricular Esquerda de medição no final de Ratos embrionárias e Neonatal

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Bioengenharia

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

In Vivo quantitativa Avaliação da Estrutura do miocárdio, função, perfusão e viabilidade Usando Cardiac Micro-Tomografia Computadorizada

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

Melhorar 2D e 3D Pele<em&gt; In Vitro</em&gt; Models Usando Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e Montagem de microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting da cartilagem e Analogs de tecido de pele utilizando um romance passiva unidade técnica de mistura para Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purificação e análise de um anticorpo monoclonal de células de ovário de hamster chinês usando um sistema de Microbioreator automatizado

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

Análise de TC quadridimensional usando registro 3D-3D sequencial

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

Imagem óptica de miócitos ventriculares isolados de urina

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Esclarecimento Primário do Fluido de Cultura Celular Colhida cho usando um separador acústico

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

Medindo kinemática de ombro 3D in vivo usando videoradiografia biplanar

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...