Harçlar Bu çalışmada, Stanford Üniversitesi, Stanford, CA, USA Tıp Fakültesi Kalp ve Damar Cerrahisi Anabilim Dalı Falk Araştırma Fonu tarafından desteklenmiştir. Tobias Deuse bir araştırma bursuyla Alman Kalp Derneği tarafından finanse edildi. Sonja Schrepfer Deutsche Forschungsgemeinschaft (DFG) (SCHR992/2-1) bir araştırma bursu aldı.

Aort Stent Dağıtım <ol><li> 550-600 g ağırlığında erkek Sprague-Dawley sıçan Harlan (Indianapolis, IN, ABD) satın alınır. House geleneksel koşullar altında sıçan, beslenen standart sıçan yemi ve su ad libitum. </li><li> Ile sıçan anestezisi izofluran (% 2) ve ketamin (25 mg / kg). Mikroskobik görünümü altında, mini laparotomi infrarenal aort maruz bir üst medyan gerçekleştirin. </li><li> Renal arter seviyesinden aşağı çatallanma, çevredeki doku abdominal aorta parçalara ayır. DİKKAT: IVC aort incelemek gerek yok. </li><li> Microclamps aort kan akışını durdurmak için kullanın. Distal kelepçe ilk proksimal kelepçe yerleştirin. </li><li> Enine küçük bir kesi ile aort açın ve heparin (200 adet) ile aort yıkayın. </li><li> Aort endotel 2-fransız Fogarty arter embolektomi kateteri (Baxter Healthcare, Deerfield, IL, USA) geçişini arındırılır. </li><li> Herhangi bir insan stent uzunluğu 8mm ve 12mm arasında büyüklüğü ve çapı 2.5mm-3mm kullanın. DİKKAT: stent çapı, pre-ve post-stent stenozu önlemek için% 10 den fazla damar çapı geçmemesi gerekir. DİKKAT: Aynı çalışma içinde stent uzunluğu açmayın. </li><li> Stent istenilen çap elde etmek için uygun bir balon basıncı kullanarak dağıtın. </li><li> Küçük aort kesi 9-0 Prolen dikişler (Ethicon, Norderstedt, Almanya) ile kapatılmıştır. </li><li> 4-0 Vicryl çalışan dikişler (Ethicon, Norderstedt, Almanya) ile katmanları karın kesi kapatın. Cilt dikişlerle hala Viacryl kullanılsa bile 7-14 gün içinde sökülmelidir. </li><li> Hayvanların içme suyu 7 gün daha intraoperatif Carprofen (6mg/KG), ve metamizol aldı. </li></ol> Optik Koherens Tomografi (OCT) Görüntüleme OCT görüntüleri M2 OCT görüntüleme sistemi (Lightlab Imaging, Inc Westford, MA, USA) ile elde edilir. ImageWire doku ışık sinyalleri toplamak için bir görüntüleme probu. ImageWire 0.006 oluşur &quot;(0,15 mm) fiber-optik çekirdek, 0.019 maksimum OD ile bir kılıf içinde&quot; (0.48 mm). <ol><li> Ile sıçan anestezisi izofluran (% 2) ve ketamin (25 mg / kg). Mikroskobik görünümü altında, infrarenal aorta ortaya çıkarmak için bir medyan yinelemek laparotomi gerçekleştirin. </li><li> Proksimal aorta ve her iki iliyak arter Kelepçe. </li><li> Aort distal ucunda enine arteriyotomi gerçekleştirin 1ml PBS 28G kateter ile yıkayın ve OCT kateter eklemek ve aort içine yönlendirmek. </li><li> Bir geri çekilme hızı saniyede 1.0 mm Motorlu geri çekilme OCT görüntüleme yapılır. </li><li> Saniyede 15 kare görüntüler elde görüntülerin bir renk look-up tablosu ile gösterilir ve dijital olarak arşivlenir. </li><li> Arteriyotomi kapatın. </li><li> 4-0 Vicryl çalışan dikişler (Ethicon, Norderstedt, Almanya) ile karın ve kesiler kapatın. Cilt dikişlerle hala Viacryl kullanılsa bile 7-14 gün içinde sökülmelidir. </li><li> Maksimum neointima oluşumu stent implantasyonu sonrası 6 hafta içinde gelişecektir. </li></ol> OCT görüntüleri analizi Ekim ölçümleri bir fare tabanlı arayüz ile Lightlab OCT görüntüleme tescilli yazılım kullanılarak yapılır. <ol><li> Sistem, bu sistem için standart kalibrasyon tekniği OCT görüntüleme tel, yansıması kalibre edin. </li><li> Trace lümen ve her biri 1.0 mm aralıklarla manuel stent kesitsel alanlar (CSA). </li><li> Lümen CSA çıkarılır stent CSA gibi plak kesit alanı hesaplayın. Stent CSA (%) bölünmesiyle plak CSA gibi yüzde plak alanını hesaplayınız. Ortalama yüzde plak alanları hesaplayın. </li></ol> OCT tekniği Geçerliliği Ekim sonuçlar histopatoloji (Şekil 1) ile ilişkilidir. Histolojik plak CSAS yukarıda açıklandığı gibi hesaplanır. Histoloji yüksek yoğunluklu ve iğ şeklinde hücreler sadece birkaç mononükleer inflamasyon hücreleri ile intimal hiperplazi ortaya koymaktadır. 6 hafta sonra, stentler tamamen neointimal granülasyon doku ile kaplıdır ve plak CSA önlemleri 1.3 ± 0.4 mm 2 2.5mm stent. <img src="/files/ftp_upload/1346/1346fig1tmb.jpg" border="0" /> Şekil 1: OCT (A) ve histolojik (B 16x büyütme) stent 6 hafta sonra dağıtım görüntüleri. OCT görüntüleri elde Plak CSA sonuçlar histopatoloji ile iyi bir korelasyon. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1346/1346fig1.jpg">buraya tıklayınız</a> .

<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>

Tüm sıçanlar standart sıcaklık, nem ve ışık koşulları altında Stanford Üniversitesi Tıp Merkezi (Stanford, Ca) hayvan bakım tesisi ev sahipliği ve yem ve su ad libitum verilmiştir. Soruşturma ABD Ulusal Sağlık Enstitüleri (NIH yayın No 85-23, 1996 revize) tarafından yayınlanan Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu nitelikteydi. Çalışma protokolü Laboratuar Hayvan Bakım Yönetim Paneli, Stanford Üniversitesi tarafından kabul edildi.

Tavşan iliak arter ve domuz koroner arter modelleri stent yerleştirilmesi 1, radyolojik ve cerrahi teçhizat gerekli bir kombinasyonu için kullanılan en sık olmakla birlikte, hayvan barınağı kapasitesi sınırlı ve satın alma maliyetleri yüksek. Sıçan stent model Sınırlamalar sıçanlar için gerekli özel olarak tasarlanmış stentlerin kullanımı, daha fazla vasküler yaralanma 2 metal-arter oranı ve tromboz (3) yapay olarak yüksek insidansı. Sıçan stent modeli, basit, ucuz, hızlı ve doğru bir preklinik model 4 . Lowe ve arkadaşları tarafından sıçan aort direkt stent ilk rapor sonra stent içi restenoz patofizyolojisi değerlendirilmesi için bu modelin 5, fizibilite ve uygunluk iyice 5,6 gösterilmiştir. Sıçan aort çapı, fizyolojik gemi mimari kesinti olmadan piyasada bulunan stentler genişleme sağlamak için yeterli. Bu tavşan ve domuz gibi trombüs oluşumu, inflamasyon, ve SMC çoğalması gibi patofizyolojik mekanizmaları, bu sıçan modelleri geliştirmek gösterilmiştir. Bu nedenle, bu modeller gerçek restenoz süreci iyi temsilleri. OCT yüksek çözünürlüklü görüntüleme teknolojisi intimal hiperplazi değerlendirmek için yararlıdır. Penetrasyon derinliği sadece 1.5-2 mm, ancak çözünürlüğü intravasküler ultrason (IVUS) 7,8 daha fazla büyüklükte bir emirdir. IVUS ile OCT karşılaştıran birçok çalışma, Ekim henüz stent implantasyonu 8-10 sonra neointimal hiperplazi değerlendirmek için tercih edilen bir teknik olduğu sonucuna varıldı. Özellikle küçük damar çapları küçük hayvanlar, yüksek çözünürlüklü OCT restenoz değerlendirme için en iyi görüntüleme yöntemidir vermektedir. Özetle, bu video gösteriyor (1) sıçan aort stent, kolayca uygulanabilir, (2) sıçan abdominal aorta stent ticari olarak üretilen stentler test etmek için uygundur ve (3) OCT görüntüleme stent boyuna izlenmesi için doğru ve zarif bir tekniktir restenoz.

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

Restenoz preklinik modeller in-stent restenoz yol ve mevcut ve gelecekteki ilaç salınımlı stentlerin optimize etmek için patofizyolojik süreçleri aydınlatmak için çok önemlidir. Sıçanlarda çeşitli antikorlar ve transgenik ve nakavt suşları mevcuttur. Sonuç olarak, sıçan in-stent restenoz için bir model pathobiological ve patofizyolojik çalışmalar için uygun olacaktır. Bu videoda, biz tam prosedür mevcut ve yüksek verimlilik stent araştırma için uygun bir sıçan stent model çukur düşer. Biz stent cerrahi prosedürü ve en zarif OCT (Optik Koherens Tomografi) tekniği kullanılarak stent restenoz değerlendirilmesi gösterecektir. Bu teknik, plak CSAS değerlendirilmesinde yüksek doğruluk sağlar (kesit alanları) ve histolojik kesitlerde, özel ve zaman alıcı gömme ve kesit alma teknikleri gerektiren koreledir. OCT görüntüleme histoloji kullanarak bir kerelik anlık görüntü kıyasla daha aynı hayvan içinde stent restenoz gelişimi boyuna izlenmesine olanak sağlar.

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

Bu video stent restenoz gelişimi pathobiological ve patofizyolojik süreçleri incelemek için ucuz ve güvenilir bir preklinik modeli nasıl kullanılacağını gösterir. OCT (Optik Koherens Tomografi) ve OCT görüntüleri analizi kullanarak in vivo izleme Boyuna da göstermiştir.

Görüntüleme-stent restenoz: Ucuz, güvenilir ve hızlı bir Preklinik Modeli

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 Görüntüleme.  Stanford University School of Medicine. Bu video stent restenoz gelişimi pathobiological ve patofizyolojik süreçleri incelemek için ucuz ve güvenilir bir preklinik modeli nasıl kullanılacağını gösterir. OCT (Optik Koherens Tomografi) ve OCT görüntüleri analizi kullanarak in vivo izleme Boyuna da göstermiştir.

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.

Ucuz mikroakışkan Cihazlar İmalat PDMS Non-plazma Yapışma

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 ...

Biyoloji

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.

Kurul, Yükleme ve Analitik ultrasantrifüjdeki Örnek Hücre Hizalama

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 Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

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.

Preklinik X-ışını bilgisayarlı tomografi Veri 3D Yazdırma Setleri

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.

Uçuş Performans Doğru ve hızlı ölçüm için geliştirilmiş Yöntemi<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.

Hızlı Analiz ve Floresans Mikroskopi Görüntüler Exploration

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.

Miyosin Ağır Zincir İmünohistokimya kullanma Sıçan kas Çapraz Bölüm kas Elyaf Nüfus Analizi İçin Hızlı Otomatik Protokolü

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.

Kardiyak resenkronizasyon sol paket şube ablasyon ve hızlı Pacing tarafından indüklenen bir zaman uyumsuz kalp yetmezliği modelindeki faydaları

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.

Yüksek işlem hacmi ölçüm larva zebra balığı kullanarak Gut Transit süre

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.