Finanzierung Die vorliegende Studie wurde von der Falk Research Fund für die Abteilung Herz-und Thoraxchirurgie an der Stanford University School of Medicine, Stanford, CA, USA unterstützt. Tobias Deuse wurde durch ein Forschungsstipendium der Deutschen Gesellschaft für Kardiologie gefördert. Sonja Schrepfer hat ein Forschungsstipendium der Deutschen Forschungsgemeinschaft (DFG) (SCHR992/2-1) erhalten.

Aortenstent Deployment <ol><li> Männliche Sprague-Dawley Ratten mit einem Gewicht 550-600 g sind von Harlan (Indianapolis, IN, USA) erworben. Haus Ratten unter üblichen Bedingungen, gefüttert Standard Rattenfutter und Wasser ad libitum. </li><li> Anesthetize Ratte mit Isofluran (2%) und Ketamin (25 mg / kg). Unter mikroskopischer Sicht, führen Sie eine obere mediane Mini-Laparotomie zur infrarenalen Aorta freizulegen. </li><li> Präparieren Sie die Bauchaorta aus dem umliegenden Gewebe, von der Ebene der Nierenarterien bis in die Bifurkation. ACHTUNG: Keine Notwendigkeit zur Aorta von der IVC zu sezieren. </li><li> Verwenden Sie microclamps der Aorta Blutfluss zu stoppen. Legen Sie die proximale Klemme erstens durch die distale Klemme gefolgt. </li><li> Öffnen Sie die Aorta mit einer kleinen queren und bündig die Aorta mit Heparin (200 Einheiten). </li><li> Die Aorta Endothel wird durch die Verabschiedung eines 2-französisch Fogarty arteriellen Embolektomie Katheter (Baxter Healthcare, Deerfield, IL, USA) entblößt. </li><li> Verwenden Sie eine beliebige menschliche Stent Größe zwischen 8 mm und 12 mm in der Länge und 2,5 mm-3mm im Durchmesser. ACHTUNG: Der Durchmesser des Stents sollte nicht mehr als der Gefäßdurchmesser um mehr als 10% auf Pre-und Post-Stent-Stenose zu vermeiden. ACHTUNG: Schalten Sie nicht Stentlänge innerhalb der gleichen Studie. </li><li> Bereitstellen des Stents unter Verwendung der entsprechenden Ballon Druck auf den gewünschten Durchmesser zu erreichen. </li><li> Der kleine Aorta Schnitt mit 9-0 Prolene Fäden (Ethicon, Norderstedt, Deutschland) geschlossen. </li><li> Schließen Sie den Bauchschnitt in Schichten mit 4-0 Vicryl fortlaufenden Nähten (Ethicon, Norderstedt, Deutschland). Die Hautnaht sollte noch innerhalb von 7-14 Tagen, auch wenn Viacryl verwendet wird entfernt werden. </li><li> Die Tiere erhielten Carprofen (6mg/kg) intraoperativ und Metamizol für 7 Tage in der Trinkwasser. </li></ol> Optical Coherence Tomography (OCT) Imaging OCT-Bilder werden mit dem M2 OCT-System (LightLab Imaging, Inc., Westford, MA, USA) erhalten. ImageWire ist ein bildgebendes Sonde an das Licht, um das Gewebe zu liefern und sammeln die Signale. Die ImageWire besteht aus 0,006 &quot;(0,15 mm) Glasfaser-Kern, in einem Mantel mit einer maximalen OD von 0,019&quot; (0,48 mm). <ol><li> Anesthetize Ratte mit Isofluran (2%) und Ketamin (25 mg / kg). Unter mikroskopischer Sicht, führen Sie eine mediane Laparotomie Redo der infrarenalen Aorta freizulegen. </li><li> Klemmen Sie den proximalen Aorta und beide iliaca. </li><li> Führen Sie eine quer Arteriotomie am distalen Ende der Aorta, mit 1 ml PBS mit einer 28G-Katheter spülen und legen Sie die OCT-Katheters und leitet es in die Aorta. </li><li> Motorisierte Pullback OCT-Bildgebung ist eine Trendwende in Höhe von 1,0 mm pro Sekunde. </li><li> Erwerben Sie Bilder mit 15 Frames pro Sekunde; Bilder sind mit einem Farb-Look-Up-Tabelle angezeigt und digital archiviert. </li><li> Schließen Sie die Arteriotomie. </li><li> Schließen Sie die Bauch-und Schnitte mit 4-0 Vicryl fortlaufenden Nähten (Ethicon, Norderstedt, Deutschland). Die Hautnaht sollte noch innerhalb von 7-14 Tagen, auch wenn Viacryl verwendet wird entfernt werden. </li><li> Die maximale Neointimabildung wird innerhalb von 6 Wochen nach Stent-Implantation zu entwickeln. </li></ol> Analysieren OCT-Bilder Oktober Messungen werden mit dem LightLab OCT-Bildgebung proprietäre Software mit einer Ratte-basierte Schnittstelle. <ol><li> Kalibrieren Sie das System auf die Reflexion des OCT-Bildgebung Draht, der die Standard-Kalibrierung Technik für dieses System ist. </li><li> Trace Lumen und Stent Querschnittsflächen (CSA) manuell auf jeder 1,0 mm Abständen. </li><li> Berechnen Plaque Querschnitt als Stent CSA aus dem Lumen CSA abgezogen. Berechnen Prozent Plaque-Bereich als Plaque CSA von Stents CSA (%) aufgeteilt. Berechnen Sie durchschnittliche prozentuale Plaque Bereichen. </li></ol> Die Validierung der OCT-Technik Oktober Ergebnisse korrelieren gut mit der Histopathologie (Abbildung 1). Histologische Plaque CSAs berechnet wie oben beschrieben. Histologisch Intimahyperplasie mit hoher Dichte von spindelförmigen Zellen und nur wenige mononukleäre Entzündungszellen. Nach 6 Wochen werden Stents komplett mit neointimale Granulationsgewebe bedeckt und die Plaque CSA Maßnahmen 1,3 ± 0,4 mm 2 in einer 2,5-mm-Stent. <img src="/files/ftp_upload/1346/1346fig1tmb.jpg" border="0" /> Abbildung 1: OCT (A) und histologische (B; Vergrößerung 16x) Bilder des Stents 6 Wochen nach der Bereitstellung. Plaque CSA Ergebnisse von OCT-Bildern erhalten korrelieren gut mit der Histopathologie. Bitte <a href="https://www.jove.com/files/ftp_upload/1346/1346fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen.

<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>

Alle Ratten wurden in der Tierstation an der Stanford University Medical Center (Stanford, CA) unter Standardbedingungen Temperatur, Luftfeuchtigkeit und Lichtverhältnisse untergebracht und wurden zur Verfügung gestellt Rattenfutter und Wasser ad libitum. Die Untersuchung des Leitfadens für die Pflege und Verwendung von Labortieren durch die US National Institutes of Health (NIH Publikation Nr. 85-23, revised 1996) veröffentlicht entsprach. Das Studienprotokoll wurde von der Verwaltungskommission Panel on Laboratory Animal Care, Stanford University zugelassen.

Obwohl das Kaninchen iliaca und das Schwein koronaren Modelle die am häufigsten für die Stentimplantation 1, eine Kombination von radiologischen und chirurgischen Geräten erforderlich ist, eingesetzt werden, ist zur Unterbringung der Tiere Kapazitäten begrenzt, und die Kosten des Erwerbs sind hoch. Einschränkungen der Ratte Stent-Modell ist der notwendige Einsatz von speziell Stents für Ratten, die Metall-Arterie Quotienten in mehr Gefäßverletzung 2, und die künstlich hohen Inzidenz von Thrombosen 3. Die Ratte Stenting-Modell ist eine einfache, kostengünstige, schnelle und genaue präklinischen Modell 4. Nach dem ersten Bericht über das direkte Stenting der Aorta von Ratten von Lowe et al. 5, Machbarkeit und Eignung dieses Modells für die Beurteilung der Pathophysiologie der In-Stent Restenose wurde gründlich 5,6 dargestellt. Der Durchmesser der Aorta von Ratten ist ausreichend, um Ausbau von kommerziell erhältlichen Stents ohne Störung der physiologischen Gefäß-Architektur ermöglichen. Es hat sich gezeigt, dass die pathophysiologischen Mechanismen, wie Thrombenbildung, Entzündungen und SMC-Proliferation, in diese Ratte Modelle zu entwickeln, wie sie bei Kaninchen und Schweinen zu tun. Daher sind diese Modelle gut Darstellungen der eigentliche Prozess der Restenose. Die OCT hochauflösenden Imaging-Technologie ist nützlich, um Intimahyperplasie zu bewerten. Die Eindringtiefe ist nur 1,5-2 mm, aber die Auflösung ist um eine Größenordnung größer als die des intravaskulären Ultraschall (IVUS) 7,8. Mehrere Studien zum Vergleich von OCT mit IVUS Schluss, dass OCT derzeit ist das bevorzugte Verfahren zur Neointimahyperplasie nach Stentimplantation 8-10 zu bewerten. Gerade in kleinen Tiere mit kleinen Gefäßdurchmesser, macht die hohe Auflösung der OCT es das beste bildgebende Verfahren für die Bewertung von Restenose. Zusammenfassend zeigt dieses Video, dass (1) Ratte Aortenstenting leicht machbar ist, (2) Ratte Bauchaorta Stenting eignet sich zur Prüfung kommerziell hergestellten Stents und (3) OCT-Bildgebung ist eine präzise und elegante Technik für Längs-Überwachung von In-Stent- Restenose.

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

Präklinische Modelle der Restenose sind unerlässlich, um die pathophysiologischen Vorgänge, die in-Stent-Restenose zu führen und bestehende und zukünftige drug-eluting stents optimieren zu entwirren. Eine Vielzahl von Antikörpern und transgenen und Knockout-Stämme sind in Ratten. Folglich würde ein Modell für In-Stent-Restenose in der Ratte sehr bequem sein pathobiologischen und pathophysiologische Studien. In diesem Video stellen wir Ihnen das gesamte Verfahren und pit-fällt einer Ratte Stent-Modell geeignet für hohen Durchsatz Stent Forschung. Wir zeigen den chirurgischen Eingriff des Stents Bereitstellung und die Beurteilung der In-Stent-Restenose mit der elegantesten Technik der OCT (Optical Coherence Tomography). Diese Technik bietet eine hohe Genauigkeit bei der Beurteilung der Plaque CSA (Querschnitt Bereiche) und korreliert gut mit histologischen Schnitten, die spezielle und zeitaufwendige Einbettung und Schneiden Techniken erfordern. OCT-Bildgebung ermöglicht ferner longitudinale Beobachtung der Entwicklung von In-Stent-Restenose innerhalb des gleichen Tieres im Vergleich zum One-Time-Snapshots mit der Histologie.

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

Dieses Video zeigt, wie eine präklinische kostengünstige und zuverlässige Modell nutzen, um pathobiologischen und pathophysiologische Prozesse in-Stent-Restenose Entwicklung zu studieren. Longitudinal in vivo-Überwachung mit OCT (Optical Coherence Tomography) und die Analyse von OCT-Bilder werden auch gezeigt.

Imaging In-Stent-Restenose: eine kostengünstige, zuverlässige und schnelle Präklinische Modell

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 Aufrufe.  Stanford University School of Medicine. Dieses Video zeigt, wie eine präklinische kostengünstige und zuverlässige Modell nutzen, um pathobiologischen und pathophysiologische Prozesse in-Stent-Restenose Entwicklung zu studieren. Longitudinal in vivo-Überwachung mit OCT (Optical Coherence Tomography) und die Analyse von OCT-Bilder werden auch gezeigt.

Serie: 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-Plasma-Bonding von PDMS für preiswerte Herstellung von Mikrofluidik-Geräte

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 ...

Forschung

Biologie

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.

Montage-, Lade-und Alignment einer analytischen Ultrazentrifuge Messkammerdirektanschluss

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.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

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.

3D Printing der präklinischen X-ray computertomographische Datensätze

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.

Ein verbessertes Verfahren für genaue und schnelle Messung der Flugleistung in<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.

Schnelle Analyse und Exploration von Fluoreszenzmikroskopie Images

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.

Ein schnelles automatisiertes Protokoll zur Muskelfaser Populationsanalyse in Rattenmuskelquerschnitte Mit Myosin Heavy Chain Immunhistochemie

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.

Vorteile der kardialen Resynchronisationstherapie in einem asynchronen Herzinsuffizienz Modell induziert durch linke Bundle Branch Ablation und schnellen Tempo

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.

Hochdurchsatz-Messung der Darm Transitzeit mit Larven Zebrafisch

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.