Financiamiento El presente estudio fue financiado por el Fondo de Investigación Falk para el Departamento de Cirugía Cardiotorácica en la Universidad de Stanford Escuela de Medicina de Stanford, California, EE.UU.. Tobias Deuse fue financiado por una beca de investigación de la Sociedad Alemana cardíaco. Sonja Schrepfer ha recibido una beca de investigación de la Deutsche Forschungsgemeinschaft (DFG) (SCHR992/2-1).

Despliegue del stent aórtico <ol><li> Ratas Sprague-Dawley de peso 550-600 g son comprados a Harlan (Indianapolis, IN, EE.UU.). Casa ratas bajo condiciones convencionales, alimentados con comida de rata estándar y agua ad libitum. </li><li> Anestesiar la rata con isoflurano (2%) y ketamina (25 mg / kg). Bajo visión microscópica, realizar una media superior de la mini-laparotomía para exponer la aorta infrarrenal. </li><li> Disecar la aorta abdominal del tejido circundante, desde el nivel de las arterias renales hasta la bifurcación. PRECAUCIÓN: No hay necesidad de diseccionar la aorta de la IVC. </li><li> Use microclamps para detener el flujo sanguíneo aórtico. Coloque la pinza proximal en primer lugar, seguido de la pinza distal. </li><li> Abra la aorta con una incisión transversal pequeña y al ras de la aorta con la heparina (200 unidades). </li><li> El endotelio aórtico es despojado por el paso de un catéter de 2-francés Fogarty embolectomía arterial (Baxter Healthcare, Deerfield, IL, EE.UU.). </li><li> Utilice cualquier tamaño humano stent entre 8 mm y 12 mm de longitud, y de 2,5 mm-3mm de diámetro. PRECAUCIÓN: El diámetro de la endoprótesis no debe exceder el diámetro de los vasos más de un 10% para evitar la estenosis pre-y post-stent. PRECAUCIÓN: No cambie la longitud del stent dentro del mismo estudio. </li><li> Desplegar el stent utilizando la presión del balón apropiado para lograr el diámetro deseado. </li><li> La incisión pequeña aórtica se cierra con suturas de Prolene 9-0 (Ethicon, Norderstedt, Alemania). </li><li> Cerrar la incisión abdominal en capas con suturas de Vicryl 4-0 en ejecución (Ethicon, Norderstedt, Alemania). La sutura de la piel aún debe ser retirado dentro de 7-14 días, incluso cuando se utiliza Viacryl. </li><li> Los animales recibieron carprofeno (6mg/kg) durante la operación, y Metamizol durante 7 días más en el agua potable. </li></ol> Tomografía de coherencia óptica (OCT) de imágenes Las imágenes de OCT se obtienen con el sistema de imágenes M2 octubre (LightLab Imaging, Inc., Westford, MA, EE.UU.). ImageWire es una sonda de imagen para ofrecer la luz a los tejidos y recoger las señales. El ImageWire consta de 0,006 &quot;(0,15 mm) de fibra óptica de núcleo, dentro de una funda con un diámetro exterior máximo de 0,019&quot; (0,48 mm). <ol><li> Anestesiar la rata con isoflurano (2%) y ketamina (25 mg / kg). Bajo visión microscópica, realizar una laparotomía mediana rehacer para exponer la aorta infrarrenal. </li><li> Abrazadera de la aorta proximal y ambas arterias ilíacas. </li><li> Realizar una arteriotomía transversal en el extremo distal de la aorta, a ras de 1 ml de PBS utilizando un catéter de 28G e inserta el catéter octubre y lo remitirá a la aorta. </li><li> Motorizados retirada octubre de imágenes se realiza a una velocidad de retroceso de 1,0 mm por segundo. </li><li> Adquirir imágenes a 15 fotogramas por segundo, las imágenes se muestran con un color look-up table y archivados digitalmente. </li><li> Cierre de la arteriotomía. </li><li> Cierre el abdomen y las incisiones con suturas de Vicryl 4-0 en ejecución (Ethicon, Norderstedt, Alemania). La sutura de la piel aún debe ser retirado dentro de 7-14 días, incluso cuando se utiliza Viacryl. </li><li> La máxima formación de la neoíntima se desarrollará dentro de las 6 semanas después del implante del stent. </li></ol> El análisis de las imágenes de OCT Las mediciones de octubre se realizan utilizando el software de imágenes LightLab octubre de propiedad con una interfaz basada en la rata. <ol><li> Calibrar el sistema a la reflexión del cable octubre de imagen, que es la técnica de calibración estándar para este sistema. </li><li> Traza luz y el stent áreas de sección transversal (CSA) de forma manual en cada uno de los intervalos de 1,0 mm. </li><li> Calcular el área de la placa de la sección transversal como AT del stent resta de la CSA luz. Calcular el área de la placa por ciento como AT de la placa, dividido por AT del stent (%). Calcular promedio de áreas de placa por ciento. </li></ol> La validación de la técnica de octubre Resultados de octubre se correlacionan bien con la histopatología (Figura 1). CSA histológico placa se calculan como se describió anteriormente. La histología muestra hiperplasia de la íntima con alta densidad de células fusiformes y las células mononucleares de sólo unos pocos la inflamación. Después de 6 semanas, los stents están completamente cubiertas con tejido de granulación neointimal y la AT de la placa medidas de 1,3 ± 0,4 mm 2 en un stent de 2,5 mm. <img src="/files/ftp_upload/1346/1346fig1tmb.jpg" border="0" /> Figura 1: octubre (A) e histológicas (B, ampliación 16x) imágenes del stent 6 semanas después de la implementación. Resultados obtenidos a partir de la placa CSA imágenes de OCT se correlacionan bien con la histopatología. Por favor, <a href="https://www.jove.com/files/ftp_upload/1346/1346fig1.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 1.

<ol>
	<li>Langeveld, B., Roks, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commentary:+rat+restenosis+models:+means+for+thorough+restenosis+research.">Commentary: rat restenosis models: means for thorough restenosis research.</a> Journal of endovascular therapy. 12 (3), 343-345 (2005).</li><li>Roguin, A., Grenadier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stent-based+percutaneous+coronary+interventions+in+small+coronary+arteries.">Stent-based percutaneous coronary interventions in small coronary arteries.</a> Acute Card Care. 8, 70-74 (2006).</li><li>Post, M. J., Waltenberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+Is+Beautiful:+A+Miniature+Stent+Model.">Small Is Beautiful: A Miniature Stent Model.</a> Arterioscler. Thromb. Vasc. Biol. 27, 701-702 (2007).</li><li>Langeveld, B., Roks, A. J. M., Tio, R. A., Boven, A. J. v. a. n., Want, J. J. L. v. a. n. d. e. r., Henning, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+abdominal+aorta+stenting:+a+new+and+reliable+small+animal+model+for+in-stent+restenosis.">Rat abdominal aorta stenting: a new and reliable small animal model for in-stent restenosis.</a> Journal of vascular research. 41 (5), 377-386 (2004).</li><li>Lowe, H. C., Chesterman, C. N., Khachigian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+aortic+stenting:+toward+a+simple+model+of+in-stent+restenosis.">Rat aortic stenting: toward a simple model of in-stent restenosis.</a> The American journal of cardiology. 88 (6), 720-721 (2001).</li><li>Groenewegen, R., Harst, R. v. a. n. d. e. r., Roks, R., Buikema, R., Zijlstra, R., Gilst, R. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+angiotensin+II+and+angiotensin+II+type+1+receptor+blockade+on+neointimal+formation+after+stent+implantation.">Effects of angiotensin II and angiotensin II type 1 receptor blockade on neointimal formation after stent implantation.</a> International Journal of Cardiology. , (2007).</li><li>de Smet, B. J. G. L., Zijlstra, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+look+at+drug+eluting+stents+with+optical+coherence+tomography.">A look at drug eluting stents with optical coherence tomography.</a> European Heart Journal. 28 (8), 918-919 (2007).</li><li>Pinto, T. L., Waksman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+of+optical+coherence+tomography.">Clinical applications of optical coherence tomography.</a> Journal of interventional cardiology. 19 (6), 566-573 (2006).</li><li>Matsumoto, D., Shite, J., Shinke, T., Otake, H., Tanino, Y., Ogasawara, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neointimal+coverage+of+sirolimus-eluting+stents+at+6-month+follow-up:+evaluated+by+optical+coherence+tomography.">Neointimal coverage of sirolimus-eluting stents at 6-month follow-up: evaluated by optical coherence tomography.</a> European Heart Journal. 28 (8), 961-967 (2007).</li><li>Kawase, Y., Suzuki, Y., Ikeno, F., Yoneyama, R., Hoshino, K., Ly, H. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonuniform+rotational+distortion+between+mechanical+IVUS+and+OCT+using+a+phantom+model.+Ultrasound+in+medicine+&amp;amp;+biology.">Comparison of nonuniform rotational distortion between mechanical IVUS and OCT using a phantom model. Ultrasound in medicine &amp;amp; biology.</a> 33 (1), 67-73 (2007).</li></ol>

Todas las ratas fueron alojadas en las instalaciones de cuidado de los animales de la Stanford University Medical Center (Stanford, CA), bajo condiciones normales de temperatura, humedad y condiciones de iluminación, y se les proporcionó comida de rata y agua ad libitum. La investigación conforme a la Guía para el Cuidado y Uso de Animales de Laboratorio publicado por el Instituto Nacional de EE.UU. de la Salud (NIH publicación N º 85-23, revisada en 1996). El protocolo fue aprobado por el Panel Administrativo de Atención de Animales de Laboratorio, Universidad de Stanford.

A pesar de la arteria ilíaca conejo y los modelos de cerdos de las arterias coronarias son los más frecuentemente utilizados para la colocación del stent 1, una combinación de material quirúrgico y radiológico se requiere, la capacidad de alojamiento de animales es limitada, y los costos de compra son altos. Limitaciones del modelo de rata stent es necesario el uso de stents diseñados específicamente para las ratas, la relación de metal a la arteria que resulta en más lesiones vasculares 2, y la incidencia artificialmente elevado de trombosis 3. El modelo de implantación de un stent rata es un modelo preclínico 4 simple, barata, rápida y precisa. Después de que el informe inicial del stent directo de la aorta de rata por Lowe et al. 5, la viabilidad y conveniencia de este modelo para la evaluación de la fisiopatología de la reestenosis intra-stent ha sido ampliamente demostrado 5,6. El diámetro de la aorta de rata es la adecuada para permitir la expansión de los stents disponibles comercialmente sin alterar la arquitectura del barco fisiológico. Se ha demostrado que los mecanismos fisiopatológicos, tales como la formación de trombos, la inflamación y la proliferación de SMC, se desarrollan en estos modelos de ratas como lo hacen en el conejo y el cerdo. Por lo tanto, estos modelos son una buena representación del proceso real de reestenosis. El octubre de alta resolución de la tecnología de imagen es útil para evaluar la hiperplasia de la íntima. La profundidad de penetración es sólo 1,5-2 mm, pero su resolución es de un orden de magnitud mayor que la de ultrasonido intravascular (IVUS) 7,8. Múltiples estudios comparando octubre con IVUS la conclusión de que octubre es actualmente la técnica preferida para evaluar la proliferación neointimal tras la implantación del stent 80-10. Especialmente en los animales pequeños con un diámetro de vasos pequeños, la alta resolución de octubre se hace la mejor modalidad de imagen para la evaluación de la restenosis. En resumen, este video muestra que (1) la colocación de stents aorta de rata es fácilmente factible, (2) la colocación de stents rata aorta abdominal es adecuado para las pruebas de stents fabricados comercialmente y (3) octubre de imágenes es una técnica precisa y elegante para el seguimiento longitudinal de los in-stent reestenosis.

imaging in-stent restenosis: an inexpensive, reliable, and rapid preclinical model

Los modelos preclínicos de reestenosis son esenciales para desentrañar los procesos fisiopatológicos que conducen a la reestenosis intra-stent y para optimizar el actual y futuro stents liberadores de fármacos. Una variedad de anticuerpos y de cepas transgénicas y knockout están disponibles en las ratas. En consecuencia, un modelo de reestenosis intra-stent en la rata sería conveniente para los estudios de patobiológicas y fisiopatológicas. En este vídeo, se presenta el procedimiento completo y pit-cae de un modelo de rata stent adecuado para la investigación de alto rendimiento del stent. Vamos a mostrar la técnica quirúrgica de implantación del stent, y la evaluación de la reestenosis intra-stent utilizando la técnica más elegante de octubre (tomografía de coherencia óptica). Esta técnica ofrece una alta precisión en la evaluación de la placa CSA (áreas de sección transversal) y se correlaciona bien con los cortes histológicos, que requieren técnicas especiales que consumen y el tiempo de inclusión y corte. Octubre de imagen permite además seguimiento longitudinal del desarrollo de la reestenosis intra-stent en el mismo animal en comparación con las instantáneas de una sola vez mediante histología.

Watch this Scientific Journal Video about Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model at JoVE.com

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Este video muestra cómo utilizar un modelo preclínico de bajo costo y confiable para estudiar los procesos fisiopatológicos patobiológicas y de desarrollo de reestenosis intra-stent. Longitudinal monitorización in vivo utilizando octubre (tomografía de coherencia óptica) y el análisis de las imágenes de OCT también se demuestre.

Imágenes de reestenosis intra-stent: un modelo barato, preclínicos fiable, rápida y

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

imaging-stent-restenosis-an-inexpensive-reliable-rapid-preclinical

Research

JoVE Journal

Biology

13.7K vistas.  Stanford University School of Medicine. Este video muestra cómo utilizar un modelo preclínico de bajo costo y confiable para estudiar los procesos fisiopatológicos patobiológicas y de desarrollo de reestenosis intra-stent. Longitudinal monitorización in vivo utilizando octubre (tomografía de coherencia óptica) y el análisis de las imágenes de OCT también se demuestre.

Video: Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

No plasmáticos de unión de PDMS para la Fabricación de bajo costo de los dispositivos de microfluidos

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Investigación

Biología

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Concentración, la carga, y la alineación de una célula de ultracentrifugación analítica de muestras

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Impresión 3D de Preclínica de rayos X de datos tomografía computarizada Establece

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Un método mejorado para la rápida y precisa medición de rendimiento en vuelo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Análisis rápido y exploración de microscopía de fluorescencia de Imágenes

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Un Protocolo Automatizado Rápido de Fibra Análisis Población en músculo de rata Secciones representativas Uso de la cadena pesada de miosina inmunohistoquímica

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac resynchronization therapy (CRT), and LBBB has become one of the important predictors for CRT response. The conventional tachypacing-induced HF model has several major limitations, including absence of stable LBBB and rapid reversal of left ventricular (LV) dysfunction after cessation of pacing. Hence, it is essential to establish an optimal model of chronic HF with isolated LBBB for studying CRT benefits. In the present study, a canine model of asynchronous HF induced by left bundle branch (LBB) ablation and 4 weeks of rapid right ventricular (RV) pacing is established. The RV and right atrial (RA) pacing electrodes via the jugular vein approach, together with an epicardial LV pacing electrode, were implanted for CRT performance. Presented here are the detailed protocols of radiofrequency (RF) catheter ablation, pacing leads implantation, and rapid pacing strategy. Intracardiac and surface electrograms during operation were also provided for a better understanding of LBB ablation. Two-dimensional speckle tracking imaging and aortic velocity time integral (aVTI) were acquired to validate the chronic stable HF model with LV asynchrony and CRT benefits. By coordinating ventricular activation and contraction, CRT uniformed the LV mechanical work and restored LV pump function, which was followed by reversal of LV dilation. Moreover, the histopathological study revealed a significant restoration of cardiomyocyte diameter and collagen volume fraction (CVF) after CRT performance, indicating a histologic and cellular reverse remodeling elicited by CRT. In this report, we described a feasible and valid method to develop a chronic asynchronous HF model, which was suitable for studying structural and biologic reverse remodeling following CRT.

The establishment of a chronic asynchronous heart failure (HF) model by rapid pacing combined with left bundle branch ablation is presented. Two-dimensional speckle tracking imaging and aortic velocity time integral are applied to validate this stable HF model with left ventricular asynchrony and the benefits of cardiac resynchronization therapy.

Beneficios de la terapia de resincronización cardiaca en un modelo asincrónico insuficiencia cardíaca inducida por la ablación de rama de paquete izquierdo y estimulación rápida

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Medida de alto rendimiento de tiempo de tránsito intestinal utilizando larvas peces cebra

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.