Name: coordinate mapping of hyolaryngeal mechanics in swallowing
Uploaded: 2014-05-06T15:30:00
Description: Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

Os autores reconhecem Kendrea Focht, cscd, CCC-SLP eo Instituto Trammell Evelyn de Voz e Deglutição da Universidade Médica da Carolina do Sul, para o compartilhamento de arquivos de imagem MBS utilizados para demonstrar esta metodologia. Estes dados foram coletados através de MBS apoio extramuros financiado pela Grant Número TL1TR000061 (PI: Focht) do Centro Nacional para a Promoção da Ciência Translacional e por Grant Número 1K24DC12801 (PI: Martin-Harris), do Instituto Nacional de Surdez e Outros Distúrbios de Comunicação e apoio intramural de Marcos e Evelyn Trammell Trust. Esses métodos foram originalmente desenvolvidos pelo pesquisador principal, enquanto apoiada por Grant Número F31DC011705 do Instituto Nacional de Surdez e Outros Distúrbios de Comunicação. O conteúdo é de responsabilidade exclusiva de seus autores e não representam, necessariamente, a posição oficial do Instituto Nacional de Surdez e Outros Distúrbios de Comunicação ou o National Institutesda Saúde.

1. Configurando um computador <ol><li> Para Macintosh, baixe o seguinte código aberto ou software freeware: ImageJ, MacX Video Converter Free Edition (Mac) e QuickTime (ver tabela de material / equipamento). </li><li> Para um PC, baixe o seguinte código aberto ou software freeware: ImageJ, MPEG streamclip (PC) e QuickTime (ver tabela de material / equipamento). </li></ol> 2. Preparando videoclipes <ol><li> A conversão de arquivos. Converta arquivos de vídeo em matérias. Mov para coleta d...

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	<li>Leonard, R. J., Kendall, K. A., McKenzie, S., Gon&#231;alves, M. I., Walker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Displacements+in+Normal+Swallowing:+A+Videofluoroscopic+Study.">Structural Displacements in Normal Swallowing: A Videofluoroscopic Study.</a> Dysphagia. 15, 146-152 (2000).</li><li>Molfenter, S. M., Steele, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+Variability+in+the+Deglutition+Literature:+Hyoid+and+Laryngeal+Kinematics.">Physiological Variability in the Deglutition Literature: Hyoid and Laryngeal Kinematics.</a> Dysphagia. 26, 67-74 (2010).</li><li>Pearson, W. G., Hindson, D. F., Langmore, S. E., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Swallowing+Muscles+Essential+for+Hyolaryngeal+Elevation+by+Using+Muscle+Functional+Magnetic+Resonance+Imaging.">Evaluating Swallowing Muscles Essential for Hyolaryngeal Elevation by Using Muscle Functional Magnetic Resonance Imaging.</a> International Journal of Radiation Oncology* Biology* Physics. 85, 735-740 (2013).</li><li>Pearson, W. G., Langmore, S. E., Yu, L. B., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+of+Muscles+Elevating+the+Hyolaryngeal+Complex.">Structural Analysis of Muscles Elevating the Hyolaryngeal Complex.</a> Dysphagia. 27, 445-451 (2012).</li><li>Kim, Y., McCullough, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+hyoid+displacement+in+normal+swallowing.">Maximum hyoid displacement in normal swallowing.</a> Dysphagia. 23, 274-279 (2008).</li><li>Steele, C. 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=The+relationship+between+hyoid+and+laryngeal+displacement+and+swallowing+impairment.">The relationship between hyoid and laryngeal displacement and swallowing impairment.</a> Clin. Otolaryngol. 36, 30-36 (2011).</li><li>Logemann, J. 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=Temporal+and+Biomechanical+Characteristics+of+Oropharyngeal+Swallow+in+Younger+and+Older+Men.">Temporal and Biomechanical Characteristics of Oropharyngeal Swallow in Younger and Older Men.</a> Journal of Speech, Language and Hearing Research. 43, 1264-1274 (2000).</li><li>Pearson, W., Langmore, S., Zumwalt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Structural+Properties+of+Suprahyoid+Muscles+and+their+Potential+for+Moving+the+Hyoid.">Evaluating the Structural Properties of Suprahyoid Muscles and their Potential for Moving the Hyoid.</a> Dysphagia. 26, 345-351 (2011).</li><li>Hopkins, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+reliability+in+sports+medicine+and+science.">Measures of reliability in sports medicine and science.</a> Sports Med. 30, 1-15 (2000).</li><li>Bingjie, L., Zhang, T., Sun, X., Xu, J., Jiang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+videofluoroscopic+analysis+of+penetration-aspiration+in+post-stroke+patients.">Quantitative videofluoroscopic analysis of penetration-aspiration in post-stroke patients.</a> Neurol. India. 58, 42-47 (2010).</li><li>Webster, M., Sheets, H. D., Alroy, J., Hunt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+introduction+to+landmark-based+geometric+morphometrics.">A practical introduction to landmark-based geometric morphometrics.</a> Quantitative Methods in Paleobiology. Paleontological Society Papers. 16, 163-188 (2010).</li><li>Inamoto, Y., 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=Evaluation+of+swallowing+using+320-detector-row+multislice+CT.+Part+II:+Kinematic+analysis+of+laryngeal+closure+during+normal+swallowing.">Evaluation of swallowing using 320-detector-row multislice CT. Part II: Kinematic analysis of laryngeal closure during normal swallowing.</a> Dysphagia. 26, 209-217 (2011).</li></ol>

Este estudo demonstra a utilidade de um método que utiliza dados de coordenadas de pontos anatômicos para calcular várias medidas cinemáticas do movimento hiolaríngeo de deglutição. Confiabilidade entre avaliadores de seis avaliadores, incluindo dois avaliadores inexperientes, por coordenadas e variáveis ​​calculadas era forte (ICC&gt; 0,90). Os resultados representativos de uma amostra aleatória de adultos saudáveis ​​não disfágicos apresentaram diferenças em algumas variáveis ​​cinemáticas e...

A fase da faringe de deglutição é um processo complexo que envolve mais de vinte e músculos esqueléticos de vários elementos para transferir um bolus da cavidade oral para o esófago, protegendo as vias aéreas. Precedendo constrição faríngea, elementos do complexo hiolaríngeo (osso hióide, da laringe e estruturas associadas, incluindo o esfíncter superior do esôfago) são deslocados para converter um canal respiratório em um aparelho digestivo. A laringe é re-localizada anteriormente longe da trajetória de um bolo que se aproxima, eo esfíncter superior do esôfago é esticada aberta. Medidas cinemáticas tiradas de vídeo estudos deglutição fluoroscopia (também conhecido como MBS ou modificados bário Estudos Andorinha) são a metodologia de pesquisa primária para quantificar os vários movimentos do complexo hiolaríngeo 1. Enquanto medições vídeo quantitativa fluoroscopia são úteis para medir função de deglutição, os diferentes eixos de referênciae escalares de resultado da medição em resultados que são incompatíveis entre os vários métodos de medições cinemáticas 2. O movimento do paciente e fluoroscope sob controle clínico manual também confunde a precisão de medição deste processo fisiológico complexo. Mais importante ainda, as medidas cinemáticas não refletem necessariamente relações importantes para a avaliação da deglutição desordenada estrutura-a-função. Cinemática do hióide, em particular, foram concebidos para acompanhar o movimento em uma direção anterior ou superior em referência a um plano anatômico alinhado com as vértebras. No entanto, essa configuração não representa a linha de ação dos músculos que suspendem o hióide. Um mecanismo de duas funda de elevação hiolaríngeo na fase faríngea da deglutição foi identificado (Figura 1) 3,4. As supra-hióide compreendem o estilingue muscular anterior, e os músculos da faringe longas compreendem o posterior estilingue muscular. Estes músculos elevar vários elementos do complexo hiolaríngeo incluindo o osso hióide, da laringe, e as estruturas que formam o esfíncter esofágico superior. Coordenar o mapeamento da mecânica hiolaríngeo utiliza nove pontos anatômicos facilmente identificáveis ​​para mapear três alavancas ósseas e características dos hiolaríngeo complexos representando pontos de fixação do anterior e slings muscular posterior (Figura 2). Durante a deglutição, cada alavanca esquelético e característica do complexo hiolaríngeo está em movimento. Através da recolha de coordenadas, o sistema pode ser capturado em qualquer período de tempo. Conversão trigonométrica de coordenadas podem ser usados ​​para gerar múltiplas medições de movimento cinemático hiolaríngeo durante a deglutição. As variáveis ​​podem ser calculadas para a comparação com os resultados relatados na literatura, ou usado para gerar novas medições representam estrutura-a-função de relações de interesse. O objetivo principal deste trabalho é demonstrar um método de geração de múltiplas medições cinemáticas calculadas a partir de um único conjunto de coordenadas marco anatômicos coletados de Modificados bário (MBS) estudos. Nós documentamos a confiabilidade desse método, utilizando coeficientes de correlação intraclasse para determinar a confiabilidade entre avaliadores de 6 investigadores diferentes, incluindo um especialista, três avaliadores com experiência, e dois novatos. A partir dos resultados cinemáticos, diferenças na deglutição mecânica por consistência bolus são avaliadas. Finalmente, a questão proposta por Molefenter e Steele sobre a importância do eixo de referência utilizado para medir o movimento hióide é abordada. Para abordar esta questão, comparar as medidas de excursão hióide em referência às vértebras e em referência à mandíbula, calculado a partir do mesmo conjunto de coordenadas para os dois tipos de bolus. Se estes dois métodos de medição do movimento hióide representam a mesma estrutura para funcionar relaçõesquadril, em seguida, os resultados devem ser fortemente correlacionado. Para este estudo, 40 estudos vista MBS laterais foram selecionados aleatoriamente a partir de uma coleção de 139 estudos normais no âmbito de um protocolo de pesquisa aprovado pelo Conselho de Revisão Institucional da Universidade Georgia Regents e em colaboração com o Instituto de pesquisa Trammell Evelyn para voz e da deglutição, da Universidade de Medicina de Carolina do Sul. Para demonstrar a utilidade deste método, dez variáveis ​​que caracterizam cinemática hiolaríngeo foram calculados a partir do mesmo conjunto de dados de coordenadas (Tabela 1). Sete destas medidas calculadas previamente utilizado na literatura, incluindo: anterior e medições de distância hióide superiores em referência às vértebras 5; anterior e deslocamento hióide superior, como uma proporção do comprimento em C2-4, também em referência às vértebras 6; movimento superior da laringe, em referência às vértebras 7; approximat hiolaríngeoião 1; e excursão máxima hióide em referência às vértebras 1. Além disso, três novas medições foram calculados: o encurtamento da faringe aproximação dos anexos do músculo palatopharyngeus, elevação laríngea seguindo uma linha de ação que representa a stylopharyngeus e excursão hióide aproximam os anexos dos músculos supra-hióideos 4,8. Uma cabeça de especialistas e anatomista pescoço (WP), três investigadores com experiência de tomar medidas limitadas (CJ, SR, TT), e dois investigadores iniciantes (RS, JT) obteve dados de coordenadas de mapeamento utilizando o protocolo descrito abaixo. O especialista (WP) treinou os três avaliadores com experiência, e estes, por sua vez treinou os dois avaliadores inexperientes. Confiabilidade entre avaliadores de coordenar dados e resultados calculados a partir de coordenadas por assunto foi determinada pelo coeficiente de correlação intraclasse 9. Dois testes t de cauda foram realizados em cada uma das variáveis ​​para determinar statisticalldiferenças significativas em y tipos de bolus. Um coeficiente de correlação de Pearson e um coeficiente de determinação foram utilizados para avaliar a concordância entre os resultados de excursão hióide calculados com as vértebras como um eixo de referência contra excursão hióide com a mandíbula como um eixo de referência para a 5 ml andorinha-líquido fino e 5 ml andorinha pudim.

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coordinate mapping of hyolaryngeal mechanics in swallowing

Caracterizando movimento hiolaríngeo é importante para a investigação da disfagia. Métodos anteriores exigem várias medições para obter uma medição cinemática enquanto coordenar o mapeamento da mecânica hiolaríngeo usando Modificado bário Swallow (MBS) usa um conjunto de coordenadas para calcular várias variáveis ​​de interesse. Para fins de demonstração, dez medições cinemáticas foram gerados a partir de um conjunto de coordenadas para determinar as diferenças em engolir dois tipos de bolus diferentes. Os cálculos das excursão hióide contra as vértebras e da mandíbula são correlacionados para determinar a importância de eixos de referência. Para demonstrar coordenar metodologia de mapeamento, 40 estudos MBS foram selecionados aleatoriamente a partir de um conjunto de dados de indivíduos normais saudáveis, sem comprometimento da deglutição conhecida. A 5 ml bolus de líquido fino e de 5 ml andorinhas pudim foram medidos a partir de cada disciplina. Nove coordenadas, mapeando a base do crânio, mandíbula, vértebras e elementos do hyocomplexo de laringe, foram registradas nos quadros de excursão mínimo e máximo hiolaríngeo. As coordenadas foram convertidos matematicamente para dez variáveis ​​da mecânica hiolaríngeo. Confiabilidade entre avaliadores foi avaliada pelo coeficiente de correlação intraclasse (ICC). Testes t bicaudal foram utilizados para avaliar diferenças na cinemática por bolus viscosidade. Medições de excursão hióide contra diferentes eixos de referência foram correlacionados. Confiabilidade entre avaliadores entre seis avaliadores para as coordenadas 18 variou de ICC = 0,90-0,97. A ardósia de dez medidas cinemáticas foi comparado por assunto entre os seis avaliadores. Um outlier foi rejeitada, ea média dos escores de confiabilidade restantes foi ICC = 0,91, 0,84-0,96, IC 95%. Bicaudal t-testes com correções de Bonferroni comparando dez variáveis ​​cinemáticas (5 ml de líquido fino andorinhas vs 5 pudim ml) apresentaram diferenças estatisticamente significativas na excursão hióide, movimento laríngeo superior e faringe shortening (p &lt;0,005). Correlações de Pearson das medidas de excursão hióide de dois eixos diferentes de referência foram: r = 0,62, r 2 = 0,38, (líquido-fina); r = 0,52, R 2 = 0,27, (pudim). Obtenção de coordenadas marco é um método confiável para gerar múltiplas variáveis ​​cinemáticas a partir de imagens de vídeo fluoroscopia úteis em pesquisa disfagia.

Os coeficientes de correlação intraclasse (ICC) de coordenadas coletadas por seis investigadores que analisaram de forma independente 80 arquivos de vídeo (fluoroscopia dois ensaios bolus de 40 indivíduos) variou de ICC = 0,90-0,97. A desagregação do ICC de de coordenadas pelo grupo é a seguinte: as coordenadas N º 1 - 5 (elementos esqueléticos em excursão mínimo hiolaríngeo) média = 0,93, 0,91-0,95, IC 95%; coordena º 6 - 9 (complexo hiolaríngeo na excursão mínimo hiolaríngeo) média = 0,94, 092-,96, ...

Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Coordenar o mapeamento é um método de documentar características marcantes da biomecânica hiolaríngeo na fase faríngea da deglutição. Esta metodologia utiliza um software de análise de imagem para gravar as coordenadas de pontos anatômicos. Estas coordenadas são importados para uma macro de excel e traduzido em variáveis ​​cinemáticas de interesse úteis na investigação de disfagia.

Coordenar Mapeamento de hiolaríngeo Mecânica em engolir

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

coordinate-mapping-of-hyolaryngeal-mechanics-in-swallowing

Research

JoVE Journal

Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Guia de Cateterismo Fio Assistida e injeção de corante colorido para o Mapeamento de Vascular monocoriônicas placentas gêmeas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

Medicina

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

Alta Resolução Mapeamento endocárdico e epicárdico óptico em um modelo de Ovinos de Stretch Induzida por Fibrilação Atrial

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Mapeamento de heterogeneidade de expressão da proteína em tumores usando imunofluorescência Quantitative

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Isolamento livre-Label e enriquecimento de células através de contato dieletroforese

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

Mapeamento MRI de Cerebrovascular Reatividade via gás para inalação Desafios

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Modelos cirúrgicos de refluxo gastroesofágico com Ratos

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Avaliação eletrofisiológica de murino átrios com mapeamento óptico de alta resolução

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Mapeamento quantitativo de ventilação específica no pulmão humano usando ressonância magnética de prótons e oxigênio como agente de contraste

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Análise de alto throughput de dados de mapeamento óptico usando o ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

Encurtamento sarcomere de Cardiomiócitos Derivados de Células-Tronco Pluripotentes usando Proteínas Sarcomere marcadas por fluorescentes.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...