The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

NOT: Bu protokol açıklanan tüm hayvan ilgili prosedürler önceden Duke Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (DUIACUC) tarafından onaylanmıştır. 1. Kanser Hücre Kültürü ve Enjeksiyon <ol><li> Floresan etiketli metastatik kanser hücrelerini Yetiştirmek (örn insan MDAMB231-GFP meme kanseri hücreleri, Dr. Patricia Steeg, NCI ve YFP etiketli fare sarkomu hücreleri, Dr David Kirsch, Duke Üniversitesi Tıp Merkezi&#39;nde Radyasyon Onkolojisi Bölümü&#39;nden hediye hediye) Uygun bir kültür ortamında (örneğin, Dulbecco Modifiye Eagle Ortamı,% 10 fetal büyükbaş hayvan serumu ve% 1 penisilin / streptavidin ile (DMEM)), 37 ° C &#39;de yaklaşık olarak% 90, birbirine karışana kadar. </li><li> Hücreleri Trypsinize PBS ile onları 2 kez yıkayın, saymak, ve sonra bir 27 G iğne ile bir şırınga kullanarak, hayvan başına 5 milyon hücre izofluran-anestezi 10 haftalık dişi çıplak sıçanların kuyruk damarlar içine enjekte edilir. Cerrahi düzey anesthesia ayak tutam tepki eksikliği tarafından doğrulanmadı. </li></ol> Metastaz kullanma mikroBT 2. İzleme <ol><li> Metastaz çapı çapı yaklaşık 2 mm varlığını tespit etmek için, bir mikro-CT / mikro ışınlayıcı kullanılarak haftada bir sıçan inceleyin. Daha önce açıklandığı gibi 12 mikro-BT devreye alınmıştır. <ol><li> Görüntüleme önce% 3 izofluran anestezisi Konu fareler. Ayak tutam derin anestezi onaylayın. </li><li> Anestezi başlangıcından sonra, hızla görüntüleme odasında görüntüleme beşiğine sıçan aktarmak ve% 2.5-3 bir izofluran-hava karışımı bir burun-koni aracılığıyla bağlayın. Görüntüleme beşiği harici pozisyon kontrolleri ve lazer sınırlayıcı kullanarak, kendi toraks mikroBT tarayıcının foton ışın konumlandırılmış bir şekilde beşikte sıçan konumunu ayarlayın. Gama ışınlarından araştırmacı kalkan, görüntüleme odasının kapısını kapalı olduğundan emin olun. </li><li> Yine kullanarak hayvanın konumunu kontrolrenkli video kamera. Düşük çözünürlüklü BT görüntüleme deneme sürüşü gerçekleştirin ve göğüs boşluğunun xyz boyutlarına görüş alanını ayarlamak için çıkan görüntü kullanın. </li><li> Görüntü sıçan 40 kVp bir 2 mm Al filtre kullanılarak toraks, 2.5 mA ve 7 FPS 0.008 Vokseller ve kafesine hayvan dönün. Bir hayvanın Görüntüleme en fazla 15 ya da 20 dakika sürer. Tamamen iyileşene kadar diğer hayvanların şirkete ameliyat geçirmiş bir hayvan iade etmeyin. Sternal yatma korumak için yeterli bilinci yerine kadar gözetimsiz bir hayvan bırakmayın. </li><li> Intratorasik kan damarları tarafından izah edilemez nispeten radyo-opak nesnelerin görünümünü tarafından metastaz onaylayın (Şekil 1A) </li></ol></li></ol> 3. Pencere Odası Cerrahisi <ol><li> Anestezi, vital bulgular ve kuyruk ven kateterizasyonu <ol><li> Metastatik hastalık varlığı ile hayvanları seçin. Bir karın içi d hayvan enjekte50 mg / kg pentobarbital ose. Devam etmeden önce ayak tutam cerrahi düzey anestezi onaylayın. Not: Anestezi protokolleri ilgili deney düzeneği uygun olmalıdır. Kolay yeniden doz seçeneği sunarken Pentobarbital, uzun prosedürler için derin anestezi uyarmak için, uzun etkili anestetik olarak burada seçildi. Ancak, doz aşımı hayvanların kaybı pentobarbital anestezi ile ortak bir sorundur. Pentobarbital daha büyük bir dereceye kadar otonom refleksleri koruyan Başka bir seçenek de, ancak sadece tek bir yeniden dozaj çevrimi için izin iksilazin ya da medetomidin gibi sedatif ile kombinasyon halinde ketamin olup. </li><li> Bir clipper kullanarak, metastatik hastalık vardır, ve boyun bölgesinde vücudun yan tarafında hayvanlar Tıraş. Cilt kalan tüm gevşek saç silin. Gevşek saç kaldırıldıktan sonra, kurumasını önlemek için, gözlere veteriner merhem uygulayın. NOT: Atimik çıplak sıçanlar requi artık saç olabilircerrahi işlemlerde devam etmeden önce res kaldırma. Cerrahi prosedürler ve görüntüleme müdahale edebilir gibi, iyice tüm saç kaldırmak için çok önemlidir. </li><li> 37 ° C su sirküle ısıtma yastığı yerleştirilmiş bir metal plaka üzerinde bir yatar pozisyonda hayvan Fix. Ön ve arka ekstremite bant ile plaka üzerine sabitlenir. NOT: Bu kontrol ve cerrahi ve deneysel prosedürler boyunca, bir pulse oksimetre ile kalp oranları ve arteriyel kan oksijenlenme gibi vital bulguları, kayıt yararlıdır. </li></ol></li><li> Trakeal entübasyon <ol><li> Trakea uzunlamasına kas ventral median ayrılması ardından çapraz servikal cilt kesisi, ilk, hayvanın havalandırma için bir kateter yerleştirmek yapmak için. </li><li> Trakea dorsal tarafı ile dikiş için bir geçiş oluşturmak için keskin bir forseps ile tekrarlanan açılış-to-kapama eylemini kullanın. </li><li> T trakea içine küçük bir kesi yapınO ventral yüzü, yaklaşık olarak ikinci ve üçüncü trakeal halka arasında, yarı dairesel daha fazla olamaz. Trakeal kateterin tespit sağlamak için, dorsal yüzeyi üzerinde açığa trakea yeterince uzun bir bölümü bırakın. </li><li> Trakea içine 2,5-3,0 mm &quot;Y&quot; trakeal kanül yerleştirin ve 4-0 monofilament sütür ile sıkın. Pozitif akciğer basıncını sağlamak için, kanül su 6 cm ile doludur son kanalına bağlı bir şişe basınç devirli ventilatöre bağlı olduğundan emin olun. Deneysel aksi istenen sürece akan gaz,% 100 oksijen olmalıdır. </li><li> Bir 25-27 G iğne ile bir kateter yerleştirin ve sıçan kuyruğu damarlar birine heparinli serum fizyolojik ile doldurulur, ve bant ile yerde sabitleyin. NOT: arka arkaya kuyruk damarından heparinli tuz az miktarda enjekte edilerek işlem boyunca kuyruk damarı kateterinin açıklığını sağlamak. Ayrıca, oro-entübasyon, bir trakeal rehberlik yanianestezi verilen hayvanın ağız yoluyla ve nefes borusu içine gırtlak geçen tüp, burada açıklanan traketomi edilen prosedüre de bir olası alternatiftir. Ancak, bu yöntem trakea zarar görmesini önlemek için, ve aynı zamanda yemek borusu yanlışlıkla kanül engelleyecek, özel eğitim ve deneyim gerektirir. </li></ol></li><li> Pulmoner pencere Uygulaması <ol><li> Künt diseksiyon kullanarak cilt ayırma sonra bir kesi oluşturarak, tarafından, metastatik hastalık bulunduğu göğüs tarafındaki deriyi çıkarın. Not: Cilt çıkarıldı ve dekolmanı sonraki kaldırılabilir </li><li> Bindirilirken kas (pektoralis, serratus ve latissimus dorsi) iki kat diseksiyon, ama sağlam interkostal kasları bırakarak devam edin. Tipik olarak, iki bitişik kaburga kısımları bertaraf eder, çap olarak yaklaşık 1.5 cm göğüs boşluğunda bir delik oluşturur. İdeal olarak, altıncı ve yedinci r bölgenin perforasyon bulunibs. </li><li> Osteotomi: <ol><li> Akciğer yüzeyine kanama ve zararı en aza indirmek için, sıkı kesme sırasında bir dişli cerrahi forseps ile kesilecek kaburga tutun. Cerrahi makas kullanılarak, dış işaret kalan kaburga kemiği sivri bir kenar bırakan, yaklaşık 45 ° &#39;lik bir açı ile, ilk kaburga medial yan kesti. </li><li> Daha sonra, yeniden akciğer yüzeyine zarar görmesini önlemek için, dışarı doğru bakan kabartma kemikten sivri bir kenar bırakan, benzer bir şekilde, kaburga kemiği yanal kenarını. </li><li> Daha sonra interkostal kasları kesmek ve kesilmiş parça kaldırın, komşu kaburga için işlemi tekrarlayın. Bu prosedür sırasında, akciğer yüzey ve göğüs kafesi arasında mekanik bir etkileşim en aza indirecek bir şekilde akciğer basınç sağlar. Uygun ventilatöre ilham basıncını düzenleyerek bunu yapın. </li></ol></li><li> Pencerenin yerleştirilmesi: <ol><li> Bir coverglass oluşan bir özel yapılmış akciğer penceresini takınbir pleksiglas soketi (Şekil 1B) eklenmiş. , Yapıştırma ya da vakum yağ çok az miktarda uygulanarak sokete pencere takın. Yüzey metastazları yakın pencerenin merkezine yer almaktadır bir şekilde pencereyi yerleştirin. Gerektiği taktirde ilgili tarafına hafifçe delik genişletilirse, pencerenin merkezi Mikrometastaz getirmek için eklenen bir delik ayarlayın. NOT: Plevral yüzeyde Metastatik hastalık çatlaklar boyunca ağırlıklı olarak görünen akciğer yüzeyinin aksi pembe-to-somon renkli sağlıklı parçalarının içinde açıkça tanınabilir beyaz noktalar veya alanlar olarak tespit edilebilir. Mikrometastazlar plevra dış diğer alanlarda oluşabilir iken, incelenen hücre çizgileri hemen hemen her zaman metastatik hastalık radyolojik tespit edilebilir kez delinmiş alanda mikrometastazları yüzey görüntüler. </li><li> Perforasyon içine soketi takarken, ve l viseral plevra ile doğrudan temas oluşturduktan sonraung, 4.0 monofilament sütür (Şekil 1C) kullanarak, çevredeki interkostal kas pencere çerçevesinin kenarlarını dikin. Kaçmak ve bir mühür oluşturmak için artık havayı yardımcı ventilatöre ilham basınç hafif bir artış kullanın. Not: sıçanların solunum hızı 70 ile 90 bpm arasında solunum hızını ayarlamak için tavsiye vb anestezi, heyecan veya anksiyete durumu, solunan havanın oksijen konsantrasyonu (FiO2), durumuna bağlı olarak, büyük ölçüde değişebilir. ilham basıncı dikkatle ayarlanmalıdır ve yaklaşık fazla ayarlanmalıdır. 8 cm H2O (0.6 mm Hg), akciğer yüzeyine zarar görmesini önlemek için. </li></ol></li><li> Flöresanlı bir mikroskop altında, bir termostatik (elektrik) ısıtma battaniyesi üzerine yerleştirilen bir çelik plaka ile Z-yönlü hareketi (Şekil 1D) ortadan kaldırmak için tasarlanmıştır özel olarak tasarlanmış süzgeç hayvan yerleştirin. Hayvanın vücudu kontrolrektal termistör kullanarak sıcaklığı. Hayvan süzgeç vidalarını ayarlayın ve ventilatöre ilham basıncı yanal hareket optimal kontrolü sağlamak için. Not: ikili, dorso-ventral ve kranyokaudal: memelilerde Doğal solunum akciğer hareketi ve göğüs uzantısı üç yön içerir. Mümkün olduğunca doğal bir solunum hareketi korumak için, gerekli ölçüde Z yönlü sıkıştırma en aza indirmek için önemlidir. Z-boyutlu emniyet kan akışını ve diğer parametreleri etkileyebilir eserleri tanıtmak potansiyeline sahip olduğundan, aynı hayvanda tekrarlayan ölçümler dizi boyunca sürekli koşullarını tutmak için tavsiye edilir. </li></ol></li></ol> 4. Görüntüleme ve Ölçüm Mikrosirkülasyonda Kan Akımının <ol><li> Kalp ponksiyonu ile kırmızı kan hücreleri toplamak ve daha önce tarif edildiği gibi, DII (1,1 = -dioctadecyl-3,3,3 = 3 = tetrametil-indokarbosiyanin perklorat) ile etiket &lt;sup&gt; 10. </li><li> Pencere bölmesi ameliyatı yapılır önce cam pencere için ilk geçiş yapışma etkilerini önlemek için, sıçanın kuyruk venine işaretli kırmızı kan hücrelerinin 300 ul enjekte edilir. Ven içine hava giriş başka enjeksiyon inhibe gibi, şırınga ya da sonda herhangi bir hava kabarcığı ortadan kaldırır. </li><li> Görüntü kan sıcaklığı -40 ° C soğutma çip bir mikroskop CCD kamera ile akış ve yaklaşık 100X toplam çözünürlük (10X mikroskop objektif yani, ve 10X ön kamera göz). Standart Rodamin / TRITC filtre setleri (uyarma 450-490 nm, emisyon&gt; 515 nm) kullanın. Ortaya çıkan görüntü dizilerinin gerçek kare hızı ve piksel çözünürlüğü kaydedin. Yığının başına rekor en az 200 (ideal etrafında 300) görüntüleri, akış hızı başarılı analizini sağlamak. </li><li> Tuzlu su ip yaklaşık 1 ml her bir saat enjekte edilerek hayvanda sıvı kaybı doldurun. NOT: Bir müdahale içeren deneysel ayarları, örneğinBir akciğer kanseri metastazı kan akış hızını değiştiren bir ilaç, pulmoner metastatik kanser kan akış hızı tekrarlanan ölçümler gerektirir. Bu genişletilmiş deneyler için, yeterli sıvı ile hayvan doldurmak için önemlidir. </li><li> Kuyruk venine 3N KCI 3 ml infüzyon ile hayvanlara Euthanize </li><li> Tüm kan akışı için akış hızı gri tonlu ve renkli kodlanmış haritalar 10,11 izleri yaratacak bir yayınlanan kamuya açık Matlab tabanlı bir bilgisayar algoritması kullanarak görüntü yığınlarını değerlendirin. Daha sonra, kan hücrelerinin hiçbir hareket gösterir aktif damarsal yani değerleri kapalı eşikleme sonra, örneğin Görüntü J gibi ticari veya kamuya açık görüntü analiz yazılımı kullanarak ortaya çıkan gri tonlu görüntüleri değerlendirir. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Bir model İntra vital mikroskopik ve hesaplama kan akışı analizi kullanılarak mikro dolaşım kan akışı ve sıçanların akciğer metastazı Diğer dinamik süreçleri, görüntü değişikliklerine mümkün olduğu gösterilmiştir. Diğer yöntemler kemirgenler açık ribcages maruz akciğerler üzerinde mikroskopi gerçekleştirmek için var iken, bu model aynı zamanda bir kapalı göğüs ortamda bir göğüs duvarı perforasyon ile görüntü pulmoner metastazlar için ilk. Bu yöntemle kullanarak, fizibil...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

Katı tümörlerde damar kıvrımlarında daha fazla derece ve daha yüksek intervasküler mesafeleri 13 gösteren normal kan akımının önemli ölçüde farklıdır bilinmektedir. Normal akciğer mikro (Şekil 2A, üst panel) ile karşılaştırıldığında duruma göre, deneysel akciğer, meme kanseri ve sarkom metastazlarında kan akışı parçalar düzensiz şekiller ve geniş intervasküler boşluklar (Şekil 2A, iki alt paneller) sahiptir. Bir önceki çalışmada, ...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Sıçan Pulmoner METASTAZLARINDA Mikrosirkülasyonda Kan Akışı Hızının Otomatik Ölçüm

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K Görüntüleme.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Video: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Fare Pulmoner Endotelyal Yüzey Katman in vivo ölçümü olarak

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Tıp

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Sigara Duman maruz kalan Fare Akciğer Amfizem ve Küçük Havayolu Yaptırma otomatik ölçüm

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Artan Pulmoner Arter Pulsatif Akış Piglets içinde Hipoksik Pulmoner Hipertansiyon Artırır

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Preterm Yenidoğan Transkutanöz Mikrosirkülasyonda Görüntüleme

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

Flow-Aracılı brakiyal dilatasyonu ve Sıçanlarda Yüzeyel Femoral Arter Ultrason Değerlendirmesi

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Sıçanlarda Karın Aort Konstrüksiyon sayesinde Kardiyak Yaptırma Bir Model

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Subklaviyan ven ponksiyon kan örnek fareleri koleksiyonunda alternatif bir yöntem olarak

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

Sıçanlarda pulmoner hipertansiyon bir Model olarak Monocrotaline ya da Sügen ile birlikte sol Pneumonectomy

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

Eşzamanlı elektrokardiyografi kayıt ve invaziv kan basınç ölçüm sıçanlarda

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...