NIH concede R01 HL085369.

1. La preparación avanzada de soluciones de reserva <ol><li> Se preparan dos soluciones estándar de solución de Tyrode (16x) de antemano en agua desionizada y almacenarlas a 4 º C: <ol class="lalpha"><li> Archivo I (119.872 g / L NaCl, 3,056 g / L de CaCl 2 (2H 2 O), 5,6 g / L KCl, 2,6274 g / L NaH 2 PO 4, 3,408 g / L MgCl 2 (6H 2 O), (Fisher Lawn científica, Feria, NJ)); </li><li> Archivo II (26,88 g / L NaHCO3, (Fisher Scientific, Fair Lawn, NJ)). </li></ol></li><li> Preparar soluciones madre de los tintes fluorescentes. Para evitar la congelación y descongelación repetida, que almacenan el 30 alícuotas de ambos colorantes a -20 ° C, lo cual es suficiente para un experimento: <ol class="lalpha"><li> Sensibles al voltaje tinte RH237 (Invitrogen, Carlsbad, CA) solución de reserva, 1,25 mg / ml en dimetilsulfóxido (DMSO, Sigma, St. Louis, MO); </li><li> Indicador de calcio Rhod-2AM (Invitrogen, Carlsbad, CA) de la solución madre, 1 mg / ml de solución de DMSO. </li></ol></li><li> Prepare la excitación-contracción blebbistatin desacoplador solución madre (Bioscience Tocris, St. Louis, MO, 2 mg / ml de solución en DMSO) con antelación y almacenar el blebbistatin disuelto a 4 ° C. </li></ol> 2. Preparar las soluciones de perfusión y la configuración experimental 7 <ol start="4"><li> Recién preparar la solución de Tyrode de 2L (128.2mM NaCl, CaCl2 1,3 mm (2H 2 O), 4.7mm KCl, 1.05mM MgCl 2 (6H 2 O), 1.19mM NaH 2 PO 4, 20 mM NaHCO 3, 11,1 mm de D-glucosa en agua desionizada, pH = 7,35 ± 0,05). Si la solución madre se está utilizando para hacer 2 litros de solución de Tyrode (suficiente para un experimento) tomar 1750 ml de agua desionizada y mezclar con 125 ml de archivo I, 125 ml de archivo II, y 4 g de glucosa. </li><li> Encienda las dos bombas de los sistemas de perfusión. Conjunto de la bomba peristáltica (Peri-Star, WPI, Sarasota, EE.UU.) que se utiliza para la perfusión retrógrada a 40 mL / min. Establecer la otra bomba peristáltica (Cole-Parmer Masterflex bomba Peristalic L / S, Cole-Parmer Instrument Company, Vernon Hills, Illinois) que se utiliza para superfusión y regresar a la perfusión de nuevo al depósito de la celebración de 80 mL / min. </li><li> Lave el sistema de perfusión con el 70% de etanol durante 30 minutos y luego con agua desionizada 2L. </li><li> Una vez que toda el agua desionizada es evacuado de la cámara, hacer circular la solución de Tyrode y pasarlo por un filtro de 5 micras (Millipore, Billerica, MA, EE.UU.). Caliente la perfusión a 37 ° C con una camisa de agua y recirculación (ThermoNESLAB EX7, Newtown, EE.UU.) y oxigenar la perfusión por burbujeo de O 2 / CO 2 (95% / 5%) de gas en la solución. Monitorear el pH de la solución con un pH-metro (Oakton Instruments, Vernon Hills, IL) y ajustar la velocidad de O 2 / CO 2 burbujas para mantener el pH a 7,35 ± 0,05. Continuar el seguimiento y la temperatura del pH durante el experimento. </li><li> El aparato de doble mapeo óptico se compone de dos cámaras Ultima Micam-L CMOS (SciMedia, Costa Mesa, CA) que tienen alta resolución espacial (100x100 píxeles, 230 ± 20 m por píxel) y la resolución temporal (1000-3000 frames / seg). Fijar un filtro pasa-banda (590 ± 15 nm, Thorlabs, Newton, Nueva Jersey) frente a la cámara de imágenes de calcio designado, mientras que, un filtro de paso largo (&gt; 700 nm, Thorlabs, Newton, Nueva Jersey), debe ser colocado en delante de la cámara de tensión imágenes designado. Las cámaras están dispuestos perpendicularmente entre sí por un titular, que contiene un espejo dicroico (635 nm de corte, Omega óptico, de Brattleboro, VT). Inmediatamente debajo del soporte de la cámara dual existe un objetivo (Nikon Nikkor 55 mm 1:1,4 235.052), que se centra la emisión de luz que viene del corazón en el espejo dicroico. La distancia de trabajo es de aproximadamente 3 cm. La luz de excitación es generada por una lámpara halógena (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) y se transmite a través de un filtro de calor, velocidad y filtro pasa banda (520 ± 45 nm). Una guía de luz flexible dirige la luz de paso de banda filtrada en la preparación, y una velocidad de obturación se utiliza para asegurar que la preparación se expone a la luz hasta la adquisición de imágenes para evitar photobleaching de los tintes. </li><li> Prepare Ag / AgCl 2 electrodos de estimulación y detección de antemano y los instala en la cámara antes de poner el corazón. Asegúrese de que los amplificadores y los filtros se ajustan a los niveles apropiados. </li></ol> 3. La cosecha del corazón de ratón, canular, y establecer la perfusión Langendorff <ol start="10"><li> Anestesiar el ratón con ketamina / xilacina (ketamina, 80mg/kg de peso corporal, la xilazina, 10 mg / kg de peso corporal) y heparina (100 unidades) por inyección intraperitoneal. Asegurar un nivel adecuado de anestesia por la falta de reflejo de dolor. </li><li> Después de una incisión en la mitad del esternón, eliminar rápidamente el corazón y lavarlo en oxigenada (95% O 2, 5% CO 2), a temperatura constante (37 ± 1 ° C) de Tyrode solución. </li><li> Utilizando un microscopio de disección, A identificar rápidamente la aorta y hacer un corte limpio a través de la aorta ascendente por debajo de la arteria subclavia derecha. Una breve sección de la aorta se conecta a un encargo de calibre 21 con una punta de la cánula quemado. 4-0 de negro trenzado sutura de seda (Corporación de Especialidades Quirúrgicas, Reading, PA) se utiliza para fijar el corazón en la cánula. Después de la canulación, el corazón es retrógrada y perfundidos superfused con Tyrode. La velocidad de perfusión retrógrada se ajusta en el rango de 5.2 mL / min para mantener la presión aórtica entre 60 y 80 mmHg (transductor de presión, World Precision Instruments Inc. (WPI), Sarasota, EE.UU.; puente Amplificador TBM4M, WPI, Sarasota, EE.UU.). </li><li> Después de que el corazón se cánula, los pulmones, el timo y el tejido graso se diseca y se retira. </li><li> El corazón aislado se fija (Herramientas Artes Ciencias) en el vértice de la parte inferior de la cámara de perfusión (recubierto Sylgard) para evitar que la corriente inducida por el movimiento. Los apéndices auriculares derecho e izquierdo también se extendía y se fija (Herramientas Artes Ciencia, Inc, Foster City, CA) a la parte inferior de la cámara, lo que proporciona un área máxima de medición óptica de las aurículas. ¡Muy importante! Un tubo de silicona que se introduce en el ventrículo izquierdo a través de las venas pulmonares y se fija con sutura de seda de tejido conectivo cerca. Esto evita la congestión y la acidificación de la solución de la perfusión atrapados en el ventrículo izquierdo, lo cual es especialmente importante después de la supresión de las contracciones ventriculares con un desacoplador de excitación-contracción (véase la Parte 4 el paso 19). </li><li> Un electrodo de encargo se coloca en la superficie del corazón para llevar a cabo la estimulación de estimulación, que se genera por el Maestro-8 (AMP Instruments Ltd, Jerusalén, Israel) o PowerLab 26T (AD Instruments, Sydney, Australia). </li><li> Una cubierta de cristal pequeño se fija en la superficie de la solución sobre el corazón para reducir los artefactos de movimiento de la solución de vibración. </li><li> Enfocar la luz de excitación en el corazón. Además, ajustar la distancia entre el aparato de doble cámara y el corazón para que la resolución máxima se obtendrá. </li><li> Apague todas las luces en la sala y comenzar con las grabaciones eléctricas PowerLab 26T. </li></ol> 4. La tensión de carga y tintes sensibles al calcio y desacoplador de excitación-contracción <ol start="19"><li> De calentamiento de 0,6 ml blebbistatin. Mezclar 0,5 ml de la blebbistatin con la perfusión en el depósito de retención. Diluir el resto de 0,1 ml de blebbistatin en 1 ml de solución de Tyrode y lentamente se inyecta (en un periodo de 20 minutos) a través de un puerto de drogas cerca de la cánula. Observe como blebbistatin reduce gradualmente el artefacto de movimiento. </li><li> Diluir 30 l de solución de la tensión de valores sensibles tinte RH237 en una solución de 1 ml de Tyrode e inyecte lentamente durante 5-7 minutos en el puerto de inyección misma blebbistatin. </li><li> 30 l de calcio indicador de Rhod-2 1:01 AM es mezclado con Pluronic F127 (Invitrogen, Carlsbad, CA, solución al 20% en DMSO) y luego se diluyen en 1 ml de solución de Tyrode y poco a poco se aplica sobre 5-10 minutos a través del puerto de inyección misma . </li><li> Esperar 5-10 minutos para blebbistatin y tintes para llegar a la membrana celular y el citosol. Continuar con el protocolo cuando el movimiento se suprime por completo. </li><li> Un seguimiento continuo de ECG durante todo el procedimiento para asegurar el normal funcionamiento eléctrico del corazón. </li><li> Comenzará grabaciones fluorescentes señal con el SciMedia de software a medida (SciMedia, Costa Mesa, CA). </li></ol> 5. Los resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> Figura 1. Arreglo experimental para la perfusión, registros eléctricos y mapeo óptico. EM = emisiones; Lp = paso largo <img alt="Figura 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> Figura 2. Preparación experimental y ejemplos de la señal registrada durante la estimulación ventricular. Izquierda: Las señales de ECG se obtienen de Ag / AgCl 2 electrodos de disco (Arriba) y un ejemplo de protocolo de estimulación S1S1 se muestra (abajo). Centro: ratón Langendorff preparación del corazón. A la derecha: Representante de los potenciales de acción óptica y señales de calcio transitorios de la aurícula (parte superior) y los ventrículos (parte inferior) se muestran. La flecha amarilla (Top) apunta a la dispersión de la señal fluorescente procedente de los ventrículos, que se ve en las grabaciones auricular. LV = ventrículo izquierdo; RV = ventrículo derecho, LA = aurícula izquierda, RA = aurícula derecha <img alt="Figura 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> Figura 3. Representante grabaciones ópticas de V m y al gato de los ventrículos del corazón del tipo de ratón salvaje. A. La preparación experimental con una serie de puntos espaciados uniformemente marcados con puntos negro cuyas grabaciones ópticas se puede ver en (C). Ejemplo B. rastreo de V m y al gato de una ubicación central en la matriz (véase el recuadro in (C)). C. V m (azul) y CAT (rojo) de la matriz de puntos espaciados uniformemente. Las señales fueron agrupadas 3x3. <img alt="Figura 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> Figura 4. Mapa de activación y conducción. A. Un ejemplo de mapa de activación de un corazón tipo de ratón salvaje con transversal (T) y longitudinal (L) direcciones indicadas por las flechas blancas. B. V señales m (Arriba) y dV / dt (abajo) que corresponden a los tres puntos de vista en A (T1, T2, T3, L1, L2, L3). <img alt="Figura 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> Figura 5. Potencial de acción y el análisis de calcio duración transitoria. A. duración potencial de acción en la repolarización del 80% (APD80) y la duración de calcio transitorio en la relajación del 80% (80 CAD) se muestran los mapas de un corazón en condiciones control (izquierda) y después de 30 aplicaciones isoproterenol nM (derecha). El color amarillo / verde en los ventrículos (derecha) indica isoproterenol acorta APD80 y 80 CAD. Ejemplo B. trazados de APD80 y 80 CAD de los ventrículos del ratón de tipo salvaje (Arriba) y en los atrios (abajo).

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No hay conflictos de interés declarado.

En este experimento se modificó el método de perfusión Langendorff mediante la adición de un pequeño tubo de silicona, que es especialmente importante después de la supresión de las contracciones ventriculares con un desacoplador de excitación-contracción. El tubo de silicona se utiliza para evitar la congestión de solución, la acidificación de la solución de perfusión, y el desarrollo de la isquemia en el ventrículo izquierdo. El corazón de ratón es muy sensible a la hipotermia, por lo que las variaciones de temperatura a través del corazón hará que las diferencias artificiales en la duración potencial de acción. En consecuencia, un sistema de calefacción se llevó a cabo en la cámara de perfusión para mantener una temperatura constante de 37 ° C durante la totalidad del experimento 8. Desde un modelo de Langendorff no retiene la inervación del corazón, es necesario considerar la incorporación de los neurotransmisores a la perfusión con el fin de investigar el tono fisiológico simpático y parasimpático 9. Además de perfusión retrógrada, además de superfusión del corazón ayuda a mantener adecuados los parámetros ambientales tales como el pH y la temperatura. En este método, el corazón Langendorff perfundidos fue colocado en posición horizontal. Una configuración de la perfusión Langendorff verticales también se puede utilizar 10, pero puede resultar en la mecánica cardíaca ligeramente diferente 11. Además de las cámaras CMOS, detectores de alternativas también están disponibles y se pueden aplicar a mapa V m y el gato al mismo tiempo 12. Aplicación de las cámaras CMOS de alta resolución espacio-temporal asegura la exactitud de las grabaciones, sin embargo, las señales ópticas de mapeo no son de una sola célula. Más bien, cada señal fluorescente proviene de cientos o miles de células, en función de magnificación óptica. La fluorescencia ventricular mucho más grande puede distorsionar las señales de dispersión óptica auricular, por lo tanto, una cuidadosa interpretación de las señales registradas ópticamente es necesario. Otra limitación de la preparación del ratón es la distorsión de la señal y el ruido inducido por la curvatura de la superficie debido al pequeño tamaño del corazón 13. Mediciones de velocidad de conducción puede ser alterada, no sólo de la curvatura del corazón del ratón, sino también de la polaridad del electrodo y los electrodos virtual. Para lograr la precisión de la velocidad de conducción, la anisotropía de la activación, y los mapas de la repolarización, enfoque correcto de la cámara en la superficie del corazón es esencial. En este método, en tiempo real ECG puede complementar la investigación óptica de la electrofisiología cardiaca. Sensibles al voltaje medio de contraste (RH237) y el indicador de calcio (Rhod-2AM) son utilizados en el protocolo debido a su rápida respuesta, la excitación similar, y los espectros de emisión de 3,7 distinta. Hay combinaciones alternativas de tintes que se pueden utilizar para medir la V m y otro gato que RH237 y 3 Rhod-2AM. Una novela de tintes sensibles al voltaje, PGHI, con un gran cambio de Stoke (&gt; 200 nm) fue encontrado para permitir una mejor m V y las señales de gato, porque la mayor separación de las longitudes de onda de emisión entre PGHI y 14 de Rhod-2AM. Mejoras en el futuro puede centrarse en la exploración de nuevas sondas fluorescentes, el desarrollo de detectores de imágenes nuevas y mejores programas informáticos de procesamiento de imágenes. Una resolución más alta y novedosa las técnicas de imagen óptico para el mapeo óptico 3D también son importantes las orientaciones futuras de mapeo óptico 5.

<table border="1"><tbody><tr><td> Químico </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> NaCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S271-1 </td></tr><tr><td> CaCl2 (2H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> C79-500 </td></tr><tr><td> KCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S217-500 </td></tr><tr><td> MgCl 2 (6H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> M33-500 </td></tr><tr><td> NaH 2 PO 4 (H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S369-500 </td></tr><tr><td> NaHCO3 </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S233-3 </td></tr><tr><td> D-glucosa </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> D16-1 </td></tr><tr><td> Blebbistatin </td><td> Bioscience Tocris, Ellisville, MO </td><td> 1760 </td></tr><tr><td> RH237 </td><td> Invitrogen, Carlsbad, CA </td><td> S1109 </td></tr><tr><td> Rhod-2AM </td><td> Invitrogen, Carlsbad, CA </td><td> R1244 </td></tr><tr><td> Pluronic F127 </td><td> Invitrogen, Carlsbad, CA </td><td> P3000MP </td></tr><tr><td> Dimetilsulfóxido (DMSO) </td><td> Sigma, St. Louis, MO </td><td> D2650 </td></tr></tbody></table>

optical mapping of action potentials and calcium transients in the mouse heart

El corazón de ratón es un modelo popular para estudios cardiovasculares, debido a la existencia de tecnología de bajo costo para la ingeniería genética en esta especie. Fenotipo cardiovascular fisiológica del corazón del ratón se puede hacer fácilmente utilizando imágenes de fluorescencia utilizando varias sondas de potencial de membrana (V m), los transitorios de calcio (CAT), y otros parámetros. Acoplamiento excitación-contracción se caracteriza por la dinámica de la acción del calcio intracelular y el potencial, por lo tanto, es muy importante planificar tanto m V y CAT simultáneamente desde la misma ubicación en el centro 4.1. Mapeo simultáneo óptica de Langendorff perfundidos corazones de ratón tiene el potencial para dilucidar los mecanismos subyacentes insuficiencia cardíaca, arritmias, enfermedades metabólicas y otras enfermedades del corazón. La visualización de la activación, la velocidad de conducción, la duración del potencial de acción y otros parámetros en una gran variedad de sitios no se puede lograr de la investigación a nivel celular, pero está bien resuelto por mapeo óptico 1,5,6. En este trabajo se presenta la configuración de los instrumentos y las condiciones experimentales para el mapeo simultáneo óptica de V m y el gato en el corazón de ratón con alta resolución espacio-temporal con el estado de la técnica de la tecnología CMOS de imagen. Consistente grabaciones ópticas obtenidas con este método ponen de manifiesto que la cartografía óptica simultánea de los corazones de ratón Langendorff perfundidos es factible y fiable.

Watch this Scientific Journal Video about Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart at JoVE.com

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Este documento detalla el procedimiento de disección, la configuración instrumental y las condiciones experimentales durante el mapeo óptico del potencial de membrana (Vm) y el calcio intracelular transitoria (CAT) en Langendorff perfundidos aislados intactos los corazones de ratón.

Mapeo óptico de potenciales de acción y transitorios de calcio en el corazón de ratón

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

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27.1K vistas.  Washington University in St. Louis. Este documento detalla el procedimiento de disección, la configuración instrumental y las condiciones experimentales durante el mapeo óptico del potencial de membrana (Vm) y el calcio intracelular transitoria (CAT) en Langendorff perfundidos aislados intactos los corazones de ratón.

Video: Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional óptico resolución microscopía fotoacústica

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Investigación

Bioingeniería

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 óptico Cartografía del corazón de conejo Langendorff perfundidos

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

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

A Cre-Lox P Enfoque de recombinación para la detección de la fusión celular En Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

La síntesis in vitro de Espaciado nativo, y colágeno fibroso Long Long segmentaria Espaciado

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Detección óptica de<em&gt; E. coli</em&gt; Bacterias por mesoporosos Silicon Biosensores

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

En Mapping in situ de las propiedades mecánicas de biopelículas por partículas de seguimiento Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparación de geles de colágeno en 3D y microcanales para el estudio de las interacciones 3D<em&gt; En Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Microscopía de fluorescencia de la hoja de luz para capturar imágenes de 4 dimensiones de los efectos de la modulación de la tensión de esquileo en el corazón de pez cebra en desarrollo

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

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.

Hoja de luz fluorescencia microscopía para el estudio del corazón 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.

Biaxiales caracterizaciones mecánicas de válvulas auriculoventricular del corazón

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