Agradecemos os drs. Raimond Winslow, Elliot McVeigh, e Patrick Helm na Johns Hopkins University para fornecer o ex vivo conjuntos de dados de pesquisa online.This foi apoiado pelo National Institutes of Health concessão R01-HL082729 e National Science Foundation concessão CBET-0933029.

1. Estimativa Orientações fibra <ol><li> Adquirir imagens de ressonância magnética e estruturais DTMRI de um coração humano adulto normal em diástole, a uma resolução de 1 mm 3. Usando ImageJ, extrair o miocárdio ventricular a partir da imagem atlas estrutural por encaixe, para cada fatia do eixo curto, fechado estrias através de um conjunto de pontos de marco colocados ao longo dos limites do epicárdio e endocárdio na fatia (Figura 2A e Figura 2B). Realizar a colocação de pontos de marco manualmente para cada fatia 10 ª na imagem. Obter os pontos marcantes para as restantes fatias através da interpolação linear dos pontos identificados manualmente, utilizando as orientações MATLAB.Reconstruct fibras do coração atlas calculando os vectores próprios primárias dos DTs na imagem DTMRI (Figura 2C). </li><li> Adquirir uma imagem da geometria do coração do paciente, em diástole cardíaca in vivo utilizando CT ou MRI. Reconstruct a geometria cardíaca do doente a partir da imagem de forma semelhante à forma como o atlas foi construído (Figura 3A e 3B). A imagem do paciente deve ser re-amostrados antes da reconstrução de tal forma que a resolução no plano é de 1 mm 2. Da mesma forma, o número de fatias para os quais pontos de referência são escolhidas manualmente, eo intervalo de fora-de-plano interpolação deve ser ajustada de modo a que a imagem do coração segmentado paciente tem uma espessura de corte de 1 mm. </li><li> Deformar a imagem do ventrículo atlas para coincidir com a geometria da imagem do paciente em duas etapas. No primeiro passo, realizar uma transformação afim com base num conjunto de pontos de 13 pontos de referência: o ventrículo esquerdo (LV) do vértice, a dois ventricular direita (VD), os pontos de inserção na base, os dois pontos de RV de inserção a meio caminho entre a base eo vértice, e quatro conjuntos de dois pontos que dividem uniformemente RV e contornos LV epicárdicas na base e intermediárias entre a base eo ápice (Figura 4A e <strong&gt; Figura 4B). No segundo passo, deformar o atlas affine transformada ventrículos adicional para coincidir com a geometria do paciente, utilizando o mapeamento difeomórfico grande deformação métrica (LDDMM) (Figura 4C). </li><li> Morph a imagem DTMRI do atlas de reposicionamento de voxels de imagem e re-orientar o DTS de acordo com a matriz de transformação da correspondência afim e o campo de deformação da transformação LDDMM. Realizar a re-orientação dos TDs usando a preservação de direções principais (PPD) do método. </li><li> Obter a estimativa das orientações de fibra a partir do paciente morphed imagem DTMRI atlas calculando o vector próprio primário do DTS (Figura 5). </li></ol> 2. Medição de erro de estimativa <ol><li> Adquirir ex vivo estrutural MR e imagens DTMR de seis normais e três falhando corações caninos, em uma resolução de 312,5 × 312,5 × 800 3 mM. Aqui, coração faiisca deve ser gerado nos caninos através ablação por radiofreqüência do ramo esquerdo, seguido de 3 semanas de tachypacing a 210 min -1. </li><li> Segmento os ventrículos do coração canino similarmente ao coração atlas humano, tal como descrito em § 1.1. Denotam ventrículos segmentados a partir de corações normais caninos como corações de 1 a 6, e os corações não segmentado de caninos como corações 7 a 9 (Figura 6). </li><li> Obter cinco diferentes estimativas de orientações ventriculares do coração de uma fibra usando cada um dos corações 2-6 como um atlas (Figura 7). </li><li> Estimar orientações de fibra para cada um dos ventrículos falência cardíaca usando 1 como o atlas (Figura 8). </li><li> Foreach ponto de dados de cada conjunto de orientação da fibra estimado, calcular o erro de estimativa como | θ θ c-a |, onde θ θ c e um são os ângulos de inclinação de uma estimad adquirido orientações de fibra nesse ponto, respectivamente. </li><li> Para cada ponto de dados de cada conjunto de orientação da fibra estimados, computar o ângulo agudo betweenestimated e adquirida direcções de fibra em três dimensões (3D), por meio de thevector produto escalar. </li></ol> 3. Mensuração dos efeitos de erro de estimativa em simulações <ol><li> De um coração, construir seis modelos, um com as orientações DTMRI-adquiridas fibra de coração 1 (referido como modelo 1), e cinco com os cinco conjuntos de dados estimados orientações de fibra (modelos 2 a 6). Para cada um dos três não cardíaca geometrias, construir dois modelos ventriculares, um com os DTMRI adquiridas orientações de fibra e outro com as orientações de fibra estimados. Aqui, a resolução espacial dos modelos, calculado em termos de comprimento da aresta média das malhas, deve ser de cerca de 600 ^ m. Denotam os modelos de insuficiência cardíaca de fibras DTMRI-adquiridas como modelos 7-9, e aqueles com Estimafibras ed como modelos de 10 a 12.In os modelos, usar a representação monodomain para descrever o tecido cardíaco, com equações que regem: </li></ol><img src="/files/ftp_upload/50125/50125eq1.jpg" alt="Equação 1" fo:content-width="2.18in" fo:src="/files/ftp_upload/50125/50125eq1highres.jpg" /> onde σ b é o tensor de condutividade a granel, que é calculado a partir dos tensores de condutividade como descrito por bidomain Potse et al 30; V m é o potencial transmembranar; C m é a capacitância de membrana específico, e I de iões é a densidade da corrente transmembranar, que por sua vez depende de V m e um conjunto de variáveis ​​de estado μ descrevendo a dinâmica de fluxos iónicos através da C membrane.For m, usar um valor de 1 μ F / cm 2. Para σ i em condições normais de modelos de coração canino, use lonnal e transversal valores de condutividade de 0,34 S / m e 0,06 S / m, respectivamente. Representar lon l pelos modelos Greenstein-Winslow iónicos do miócito ventricular canino. Diminuir os de condutividade elétrica em caninos coração modelos falência ventricular em 30% (Figura 9). <ol start="2"><li> Usando o software de pacote CARP (CardioSolv, LLC), simular ritmo sinusal, com todos os modelos. Induzir taquicardia ventricular reentrante (VT) em seis modelos de falência utilizando um protocolo de estimulação S1-S2. Escolha o tempo entre S1 e S2 para obter atividade TV sustentada por 2 segundos após o parto S2. Se VT não é induzida por qualquer tempo S1-S2, diminuir as condutividades em até 70%, até VT foi induzida (Figura 10). </li><li> Para cada simulação, calcular pseudo-ECGs tomando a diferença de potencial entre dois pontos extracelulares num banho isotrópico em torno dos corações. Colocar os dois pontos perto da base do coração, separadopor 18 cm, de tal forma que a linha que os une é perpendicular ao plano de base de ápice do septo, conforme ilustrado na Figura 10. Para cada simulação com orientações de fibra estimados, calcular a métrica como MAD </li></ol><img src="/files/ftp_upload/50125/50125eq2.jpg" alt="Equação 2" fo:content-width="2.77in" fo:src="/files/ftp_upload/50125/50125eq2highres.jpg" /> onde X é a forma de onda do ECG da simulação com as orientações de fibra de cerca de, Y é a forma de onda do ECG de thecorresponding simulação com as orientações de fibra adquiridos, X é o valor médio de X, Y é o valor médio de Y, e n é o comprimento de X e Y.

<ol>
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Esta pesquisa demonstra quantitativamente que, na ausência de DTMRI, orientações de fibra do miocárdio dos ventrículos normais e não pode ser estimada a partir de imagens in vivo das suas geometrias para utilização em simulações de electrofisiologia cardíaca. A metodologia proposta é demonstrado com dados in vivo TC, mas é igualmente aplicável a imagens de RM in vivo da geometria ventricular, dirigindo-se a falta de capacidade para adquirir directamente orienta?...

<html> A abordagem computacional está a tornar-se central para o avanço do conhecimento da função do coração na saúde e na doença. Estado-da-arte todo coração modelos de eletrofisiologia e eletromecânica estão sendo usados ​​para estudar uma ampla gama de fenômenos, como a propagação ventricular normal, arritmia, desfibrilação, acoplamento eletromecânico, e ressincronização cardíaca 1. No entanto, para a abordagem computacional para ser directamente aplicável no ambiente clínico, é imperativo que os modelos ser específicas do paciente, isto é, os modelos devem basear-se na arquitectura específica e propriedades electrofisiológicas ou electromecânicos de coração doente do paciente. Simulação com esses modelos ajudará os médicos a chegar a decisões altamente personalizados para intervenções eletrofisiológicas bem como profilaxia, reduzindo drasticamente melhorar a saúde cardíaca 2-4. content &quot;&gt; Criação de modelos cardíacos realistas requer a obtenção da estrutura e geometria da fibra de um coração do paciente. orientações de fibra determinar direcções de propagação e as distribuições de deformação eléctrica no coração, e portanto, antes de os adquirir é essencial para a modelagem cardíaco 5, 6. Com recentes avanços na imagiologia médica, é agora possível obter a geometria de um coração do paciente, a remodelação includingstructural tais como enfarte, in vivo com alta resolução usando imagem por ressonância magnética (MRI) e tomografia computadorizada (CT) tecnologias. No entanto, não há nenhuma método prático para a obtenção de uma estrutura de fibras do coração do paciente, in vivo. Diffusion tensor (DT) MRI 7, 8, a única técnica de adquirir orientações de fibras do coração intactos, não é amplamente disponível, in vivo, devido a certas limitações 9. Uma breve descrição dos esforços anteriores para traduzir DTMRI o quadro clínico pode ser encontrada elsewhere 2. Apesar de metodologias como a regra de atribuição baseada em fibra de orientações oferecer alternativas ao DTMRI, estas metodologias têm certas limitações graves 2, 10. Assim dificuldades na aquisição de estrutura de fibra cardíaca in vivo atualmente impedem a aplicação de eletrofisiológicos e eletromecânica simulações cardíacas em ambiente clínico. O objetivo desta pesquisa foi o de abordar diretamente essa necessidade. Colocámos a hipótese de que as orientações de fibras ventriculares de um coração pode ser prevista com precisão, dada a geometria do coração e um atlas, onde o atlas é um coração cuja geometria e fibra orientações estão disponíveis. Assim, usamos estado das técnicas de arte para desenvolver uma metodologia para a estimativa de orientações fibra cardíaca in vivo, e testou a hipótese em normal e não ventrículos caninos 2. A idéia central da nossa metodologia de estimativa de fibra é explorar similarities em orientações de fibras, em relação à geometria, entre corações diferentes a fim de aproximar a estrutura de fibra de um coração (alvo) para os quais as informações a geometria está disponível. No coração de nossa metodologia de estimação é o registro da geometria atlas com a geometria alvo usando mapeamento difeomórfico grande deformação métrica (LDDMM) 11, eo morphing de orientações de fibra atlas usando preservação de componentes principais (PPD) 2, 12. Diffeomorphicproperty A de garantias LDDMM que o atlas não &quot;foldover&quot; em si durante a deformação, preservando assim as estruturas anatômicas integrityof. figura 1 ilustra o pipeline de processamento de nossa metodologia. A secção de texto de protocolo § 1 descreve os vários componentes do oleoduto, demonstrando como o cálculo pode ser executado para um paciente exemplo. Os números dentro de alguns dos blocos na Figura 1 referem-se ao correspondentesubseções ao abrigo da secção § 1 º do texto do protocolo. Foi avaliado o desempenho da metodologia proposta pela quantificação do erro de estimação, e medir o efeito deste onsimulations erro de eletrofisiologia cardíaca, por simular computacionalmente locais mapas de ativação elétrica, bem como pseudo-eletrocardiogramas (ECGs pseudo-). Devido à indisponibilidade dos corações humanos, a avaliação de desempenho foi realizado utilizando corações caninos disponíveis a partir de estudos anteriores 13-15. O erro de estimativa foi calculada por meio de ângulos de inclinação 16, a tradição followingthe de histologia, onde as medições são secções angulares performedon tecidos que são cortadas paralelamente à epicardialsurface. Desde que o sentido das fibras anglebetween e plano tangente epicardial é generallysmall 17, 18, ​​a perda de informação em descrever um fiberdirection inteiramente usando o seu ângulo de inclinação é insignificante. Para o computcionais, simulações de imagem baseados em modelos foram construídos como descrito previamente 19, 20, e o tecido cardíaco nos modelos foi representada com base em técnicas matemáticas estabelecidas e os dados experimentais 21-25. Ritmo sinusal foi simulado por activação de replicação proveniente da rede de Purkinje 26, e taquicardia ventricular, por uma estimulação S1-S2 protocolo 27. Pseudo-ECGs foram computados 28 e comparados utilizando o desvio médio absoluto (MAD) métrica 29.

<table border="1"><tbody><tr><td> LDDMM </td><td> Johns Hopkins University </td><td></td><td> <a href="http://cis.jhu.edu/software/lddmm-volume/index.php" target="_blank">http://cis.jhu.edu/software/lddmm-volume/index.php</a> </td></tr><tr><td> MATLAB </td><td> Mathworks, Inc. </td><td> R2011b </td><td> <a href="http://www.mathworks.com/products/matlab/" target="_blank">http://www.mathworks.com/products/matlab/</a> </td></tr><tr><td> ImageJ </td><td> National Institutes of Health </td><td></td><td> <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> Tarântula </td><td> CAE Software Solutions </td><td></td><td> <a href="http://www.meshing.at/Spiderhome/Tarantula.html" target="_blank">http://www.meshing.at/Spiderhome/Tarantula.html</a> </td></tr><tr><td> CARP </td><td> CardioSolv</td><td></td><td> <a href="http://cardiosolv.com/" target="_blank">http://cardiosolv.com/</a> </td></tr><tr><td> Imagens caninos </td><td> Johns Hopkins University </td><td></td><td> <a href="http://www.ccbm.jhu.edu/research/DTMRIDS.php" target="_blank">http://www.ccbm.jhu.edu/research/DTMRIDS.php</a> </td></tr></tbody></table>

patient-specific modeling of the heart: estimation of ventricular fiber orientations

Específicas do paciente simulações da função cardíaca (dis), destinado a personalizar a terapia cardíaca são dificultados pela ausência de tecnologia de imagem in vivo para adquirir clinicamente orientações fibras miocárdicas. O objetivo deste projeto foi desenvolver uma metodologia para estimar orientações de fibra cardíaca em imagens in vivo de geometrias coração do paciente. Uma representação exacta da geometria ventricular e orientações de fibra foi reconstruído, respectivamente, de alta resolução, ressonância ex vivo estrutural magnética (RM) e imagens do tensor de difusão (DT) de RM de um coração humano normal, referido como o atlas. Geometria ventricular do coração do paciente foi extraído, através da segmentação semi-automática, a partir de uma in vivo tomografia computadorizada de imagem (CT). Utilizando algoritmos de transformação da imagem, a geometria ventricular atlas foi deformada para corresponder ao do paciente. Finalmente, o campo de deformação foi aplicado à fibra atlas orientatíons para obter uma estimativa de orientações de fibra de pacientes. A precisão das estimativas de fibra foi avaliada usando seis normais e três falhando corações caninos. A diferença média absoluta entre ângulos de inclinação de orientações de fibra adquiridos e estimada foi de 15,4 °. Simulações computacionais de mapas de ativação ventricular e pseudo-ECG em ritmo sinusal e taquicardia ventricular indicaram que não há diferenças significativas entre as orientações de fibra estimados e adquirido em um clinicamente observáveis ​​level.The novos insights obtidos a partir do projeto vai preparar o caminho para o desenvolvimento de paciente modelos específicos do coração, que pode ajudar os médicos no diagnóstico personalizado e decisões sobre intervenções eletrofisiológicas.

Figura 11, AC exibe visualizações simplificadas de estimativa, bem como orientações DTMRI derivados de fibra em corações normais e falhando. A análise qualitativa mostra que as orientações de fibra de cerca de alinham bem com DTMRI derivados entes. O painel D ilustra, sobreposta sobre a geometria do coração 1, a distribuição do erro de ângulos de inclinação corações normais &quot;, em média, em todos os cinco estimativas. Painel E mostra a distribuição média de erro de falha ângul...

Watch this Scientific Journal Video about Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations at JoVE.com

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Uma metodologia para estimar orientações de fibra de ventriculares em imagens in vivo de geometrias coração do paciente para a modelagem de personalizado é descrito. Validação da metodologia realizada utilizando normal e não corações caninos demonstrar que que não há diferenças significativas entre as orientações de fibra estimadas e adquirida a um nível clinicamente observável.

Paciente específico Modelagem do Coração: Estimativa de Orientações de fibra ventriculares

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

patient-specific-modeling-heart-estimation-ventricular-fiber

Research

JoVE Journal

Bioengineering

13.6K visualizações.  Johns Hopkins University. Uma metodologia para estimar orientações de fibra de ventriculares em imagens in vivo de geometrias coração do paciente para a modelagem de personalizado é descrito. Validação da metodologia realizada utilizando normal e não corações caninos demonstrar que que não há diferenças significativas entre as orientações de fibra estimadas e adquirida a um nível clinicamente observável.

Vídeo: Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

De alta resolução de fibra óptica para Microendoscopy In situ Celular imagem

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Bioengenharia

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrico Mapping Optical do Coração Coelho Langendorff-perfundidos

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Segmentação Neuronal fibra folhetos para a terapia de estimulação profunda do cérebro usando modelos interativos, paciente específico

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Microscopia de fluorescência de luz-folha para o estudo do coração murino

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxial caracterizações mecânicas das válvulas cardíacas Atrioventricular

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

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 ...

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk stratification in various cardiac disorders. Recently, 3D echocardiography became available in routine cardiology practice; however, high-quality image acquisition and subsequent analysis have a steep learning curve. The present article aims to guide the reader through a detailed 3D protocol by presenting tips and tricks and also by highlighting the potential pitfalls to facilitate the widespread but technically sound use of this important technique concerning the LV. First and foremost, we show the acquisition of a high-quality 3D dataset with optimal spatial and temporal resolution. Then, we present the analytical steps toward a detailed quantification of the LV by using one of the most widely applied built-in software. We will quantify LV volumes, sphericity, mass and also systolic function by measuring ejection fraction and myocardial deformation (longitudinal and circumferential strain). We will discuss and provide clinical examples about the essential scenarios where the transition from a conventional echocardiographic approach to a 3D-based quantification is highly recommended.

In this article, we provide a step-by-step acquisition and analysis protocol for the volumetric assessment and speckle-tracking analysis of the left ventricle by 3D echocardiography, particularly focusing on practical aspects that maximize the feasibility of this technique.

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 ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Fabricação fantasma do álcool polivinyl específico do paciente com ultrassom e contraste de raios-X para planejamento de cirurgia de tumor cerebral

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict regional aerosol delivery. Leveraging 3D printing to generate patient-specific lung models, we outline the design of a high-throughput, in vitro experimental setup for quantifying lobular pulmonary deposition. This system is made with a combination of commercially available and 3D printed components and allows the flow rate through each lobe of the lung to be independently controlled. Delivery of fluorescent aerosols to each lobe is measured using fluorescence microscopy. This protocol has the potential to promote the growth of personalized medicine for respiratory diseases through its ability to model a wide range of patient demographics and disease states. Both the geometry of the 3D printed lung model and the air flow profile setting can be easily modulated to reflect clinical data for patients with varying age, race, and gender. Clinically relevant drug delivery devices, such as the endotracheal tube shown here, can be incorporated into the testing setup to more accurately predict a device&#8217;s capacity to target therapeutic delivery to a diseased region of the lung. The versatility of this experimental setup allows it to be customized to reflect a multitude of inhalation conditions, enhancing the rigor of preclinical therapeutic testing.

We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles.

Avaliação do depoimento pulmonar regional usando modelos pulmonares impressos 3D específicos do paciente

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Quantificação da função ventricular esquerda do coração do rato, tensão do miocárdio e forças hemodinâmicas por ressonância magnética cardiovascular

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...