Finanziamento Il presente studio è stato sostenuto dal fondo di ricerca Falk per il Dipartimento di Chirurgia cardiotoracica della Stanford University School of Medicine, Stanford, CA, USA. Tobias Deuse è stato finanziato da una borsa di studio della Società Tedesca cardiaca. Sonja Schrepfer ha ricevuto un assegno di ricerca dalla Deutsche Forschungsgemeinschaft (DFG) (SCHR992/2-1).

Aortica distribuzione stent <ol><li> Maschi Sprague-Dawley di peso 550-600 g sono acquistati da Harlan (Indianapolis, IN, USA). Ratti casa in condizioni convenzionali, alimentati standard di ratto cibo e acqua ad libitum. </li><li> Anestetizzare ratto con isoflurano (2%) e ketamina (25 mg / kg). In vista microscopico, eseguire una media superiore mini-laparotomia per esporre l&#39;aorta sottorenale. </li><li> Sezionare l&#39;aorta addominale dal tessuto circostante, dal livello delle arterie renali fino alla biforcazione. ATTENZIONE: Non c&#39;è bisogno di sezionare l&#39;aorta dalla IVC. </li><li> Usa microclamps per fermare il flusso aortico sangue. Posizionare il morsetto prossimale per primo, seguito dal morsetto distale. </li><li> Aprire l&#39;aorta con una piccola incisione trasversale e lavare l&#39;aorta con eparina (200 unità). </li><li> L&#39;endotelio aortico è denudato dal passaggio di un 2-francese catetere Fogarty embolectomia arteriosa (Baxter Healthcare, Deerfield, IL, USA). </li><li> Utilizzare qualsiasi dimensione umana stent tra 8mm e 12mm di lunghezza e 2,5 mm-3mm di diametro. ATTENZIONE: Il diametro dello stent non dovrebbe superare il diametro del vaso di oltre il 10% per evitare stenosi pre-e post-stent. ATTENZIONE: Non accendere la lunghezza dello stent all&#39;interno dello studio stesso. </li><li> Distribuire lo stent con palloncino alla pressione appropriata per ottenere il diametro desiderato. </li><li> L&#39;incisione aortica piccolo si chiude con 9-0 punti di sutura Prolene (Ethicon, Norderstedt, Germania). </li><li> Chiudere l&#39;incisione addominale a strati con 4-0 punti di sutura Vicryl esecuzione (Ethicon, Norderstedt, Germania). Le suture cutanee devono ancora essere rimosse entro 7-14 giorni, anche quando Viacryl viene utilizzato. </li><li> Animali ricevuto Carprofen (6mg/kg) durante l&#39;intervento, e Metamizol per 7 giorni in più in acqua potabile. </li></ol> Optical Coherence Tomography (OCT) Imaging Immagini ottobre si ottengono con i PTOM M2 sistema di imaging (Lightlab Imaging, Inc., Westford, MA, USA). ImageWire è una sonda di imaging per fornire la luce al tessuto e raccogliere i segnali. Il ImageWire composto da 0,006 &quot;(0,15 mm) in fibra ottica core, all&#39;interno di una guaina con un diametro esterno massimo di 0,019&quot; (0,48 mm). <ol><li> Anestetizzare ratto con isoflurano (2%) e ketamina (25 mg / kg). In vista microscopico, eseguire una laparotomia mediana rifare per esporre l&#39;aorta sottorenale. </li><li> Morsetto l&#39;aorta prossimale ed entrambi arteria iliaca. </li><li> Eseguire una arteriotomia trasversale alla fine distale dell&#39;aorta, a filo con 1ml PBS utilizzando un catetere 28G e inserire il catetere ottobre e lo trasmette nell&#39;aorta. </li><li> Motorizzato pullback ottobre l&#39;imaging viene effettuata ad un tasso pullback di 1,0 mm al secondo. </li><li> Acquisire immagini a 15 fotogrammi al secondo, le immagini vengono visualizzate con una tabella di colori di look-up e archiviati in formato digitale. </li><li> Chiudere il arteriotomia. </li><li> Chiudere l&#39;addominale e incisioni con 4-0 punti di sutura Vicryl esecuzione (Ethicon, Norderstedt, Germania). Le suture cutanee devono ancora essere rimosse entro 7-14 giorni, anche quando Viacryl viene utilizzato. </li><li> La formazione si svilupperà neointima massimo entro 6 settimane dopo l&#39;impianto dello stent. </li></ol> Analizzando le immagini Ottobre Misure ottobre vengono eseguite utilizzando il software proprietario Lightlab ottobre imaging con un topo-based. <ol><li> Calibrare il sistema alla riflessione del filo ottobre imaging, che è la tecnica standard di calibrazione per questo sistema. </li><li> Traccia lume dello stent e aree delle sezioni trasversali (CSA) manualmente ad ogni intervallo 1,0 mm. </li><li> Calcolare l&#39;area trasversale della placca come CSA stent sottratto dal CSA lume. Calcolare placca zona per cento CSA placca diviso per CSA stent (%). Calcolare la media aree placca per cento. </li></ol> Validazione della tecnica di ottobre Risultati Ottobre correlano bene con l&#39;istopatologia (Figura 1). CSA placca istologici sono calcolati come sopra descritto. L&#39;istologia rivela iperplasia intimale con alta densità di cellule fusiformi e solo poche cellule mononucleate infiammazione. Dopo 6 settimane, gli stent sono interamente rivestiti con tessuto di granulazione neointimale e il CSA placca misure di 1,3 ± 0,4 mm 2 in uno stent da 2,5 mm. <img src="/files/ftp_upload/1346/1346fig1tmb.jpg" border="0" /> Figura 1: Ottobre (A) e istologiche (B, ingrandimento 16x) le immagini dello stent 6 settimane dopo la distribuzione. Risultati ottenuti da CSA placca immagini Ottobre correlano bene con l&#39;istopatologia. Si prega di <a href="https://www.jove.com/files/ftp_upload/1346/1346fig1.jpg">cliccare qui</a> per vedere una versione più grande della 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>

Tutti i ratti sono stati ospitati nella struttura cura degli animali presso la Stanford University Medical Center (Stanford, Ca), alla temperatura standard, umidità e condizioni di illuminazione, e sono stati forniti ratto cibo e acqua ad libitum. L&#39;indagine conforme alla Guida per la cura e l&#39;uso di animali da laboratorio pubblicato dal National Institutes of Health (NIH pubblicazione n ° 85-23, riveduta nel 1996). Il protocollo dello studio è stato approvato dal gruppo amministrativa per la cura degli animali da laboratorio, la Stanford University.

Anche se l&#39;arteria iliaca coniglio e maiale i modelli delle arterie coronarie sono le più frequentemente utilizzate per il posizionamento dello stent 1, una combinazione di radiologia e di attrezzature chirurgiche è richiesto, capacità di alloggiamento animale è limitata, ed i costi di acquisto sono elevati. Limiti del modello di ratto stent è necessario l&#39;uso di stent progettato specificamente per i ratti, il metallo-arteria rapporto con conseguente più danno vascolare 2, e l&#39;incidenza artificialmente elevato di trombosi 3. Il modello di stenting ratto è un 4 semplice, economico, rapido e accurato modello preclinico. Dopo la relazione iniziale di stenting diretto dell&#39;aorta ratto da Lowe et al. 5, fattibilità e l&#39;idoneità di questo modello per la valutazione della fisiopatologia della ristenosi in-stent è stato completamente dimostrato 5,6. Il diametro dell&#39;aorta ratto è sufficiente per consentire l&#39;espansione degli stent disponibili in commercio senza interruzioni dell&#39;architettura nave fisiologico. E &#39;stato dimostrato che i meccanismi fisiopatologici, come la formazione di trombi, l&#39;infiammazione e la proliferazione SMC, si sviluppano in questi modelli di ratto come fanno nel coniglio e maiale. Di conseguenza, questi modelli sono rappresentazioni bene il processo reale di ristenosi. L&#39;alta risoluzione ottobre tecnologia di imaging è utile per valutare iperplasia intimale. La profondità di penetrazione è solo 1,5-2 mm, ma la sua risoluzione è un ordine di grandezza superiore a quella degli ultrasuoni intravascolari (IVUS) 7,8. Diversi studi a confronto con ottobre IVUS concludere che ottobre è attualmente la tecnica preferita per valutare iperplasia neointimale dopo 8-10 impianto di stent. Soprattutto nei piccoli animali con diametri dei piccoli vasi, l&#39;alta risoluzione di Ottobre lo rende migliore la modalità di imaging per la valutazione della ristenosi. In sintesi, questo video mostra che (1) aortica stenting ratto è facilmente fattibile, (2) stent aorta addominale di ratto è adatto per i test stent fabbricato industrialmente e (3) Ottobre imaging è una tecnica precisa ed elegante per il monitoraggio longitudinale di in-stent ristenosi.

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

Modelli preclinici di restenosi sono essenziali per svelare i processi fisiopatologici che conducono alla ristenosi in-stent e per ottimizzare esistenti e futuri stent medicati. Una varietà di anticorpi e di ceppi transgenici e knockout sono disponibili nei ratti. Di conseguenza, un modello per la ristenosi in-stent nel ratto sarebbe conveniente per gli studi pathobiological e fisiopatologici. In questo video, vi presentiamo l&#39;intera procedura e tranelli di un modello di topo stent adatto per ricerca di alta velocità dello stent. Mostreremo la procedura chirurgica di stent, e la valutazione della ristenosi in-stent con la tecnica più elegante di OCT (Optical Coherence Tomography). Questa tecnica assicura un&#39;elevata precisione nella valutazione CSA placca (zone di sezione trasversale) e si correla bene con le sezioni istologiche, che richiedono tecniche speciali che consumano tempo e embedding e sezionamento. Ottobre di imaging consente inoltre il monitoraggio longitudinale dello sviluppo della ristenosi in-stent all&#39;interno dello stesso animale in confronto a un tempo istantanee con istologia.

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

Questo video illustra come utilizzare un modello preclinico economico e affidabile per lo studio dei processi fisiopatologici pathobiological e di sviluppo in-stent restenosi. Longitudinale monitoraggio in vivo utilizzando OCT (Optical Coherence Tomography) e l&#39;analisi delle immagini Ottobre sono anche dimostrato.

Imaging ristenosi in-stent: un poco costoso, modello affidabile, rapido e preclinici

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 Views.  Stanford University School of Medicine. Questo video illustra come utilizzare un modello preclinico economico e affidabile per lo studio dei processi fisiopatologici pathobiological e di sviluppo in-stent restenosi. Longitudinale monitoraggio in vivo utilizzando OCT (Optical Coherence Tomography) e l'analisi delle immagini Ottobre sono anche dimostrato.

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.

Non plasmatici Incollaggio di PDMS Economico per la Fabbricazione di dispositivi microfluidici

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 ...

Ricerca

Biologia

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.

Assemblaggio, carico, allineamento e di una cella analitica del campione Ultracentrifuga

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.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

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.

La stampa 3D di preclinica X-tomografia computerizzata a raggi Data Set

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 metodo migliore per precisa e rapida misurazione delle performance in volo<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.

Analisi rapida ed esplorazione di microscopia a fluorescenza Immagini

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 rapido protocollo automatizzato per Fibra muscolare Analisi Popolazione nel ratto muscolari sezioni trasversali utilizzando una catena pesante della miosina immunoistochimica

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.

Benefici della terapia di risincronizzazione cardiaca in un modello asincrono insufficienza cardiaca indotta da ablazione di ramo di pacco di sinistra e cadenza veloce

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.

High-throughput misurazione del tempo di transito dell'intestino utilizzando Zebrafish larvale

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.