Etik deyimi: Bütün deneyler Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. Hayvan bakımı kurumsal kurallarına göre oldu. (- 8 hafta eski 7, 25-30 gr) ICR dişi fareler Tel-Aviv Üniversitesi Sackler Tıp Fakültesi klimalı, ışık kontrollü hayvan tesislerinde muhafaza edildi. Vadede, hayvanlar anestezi doz aşımı ile ötenazi edildi. 1. lifli Konfokal Floresan Mikroskobu (FCFM) Kalibrasyon <ol><li> Aygıtı AÇIN. </li><li> Mikroprob (mini0 / 30) bağlayın. </li><li> Üreticinin talimatlarına göre cihazı kalibre edin. </li></ol> 2. Fareler Görüntüleme Hazırlık <ol><li> Her iki Ketaset (100 mg / kg) ve xyl-M2 (6 mg / kg) deri altından enjeksiyonu ile anestezi. Ayak-tutam tepkisizlik tarafından uygun anesthetization onaylayın. </li><li> Sırayla için kasık altındaki cildi insizyon<html> femoral arter damarları ortaya. Kesi aşağıdaki tuzlu su ile nemli kesi sitesini tutun. </li><li> Yaklaşık 30 saniye için, ılık su (çok sıcak dokunma) ile dolu bir torba (ya da bir eldiven) ile kuyruk ısıtın. Tuz veya kemoterapötik ajanın ya kuyruk damarına bir iğne (30 g, 1/2 inç) sokulması ve buna 1 ml şırınga takılarak FITC dekstran (kontrast bir madde) verilmesi için bir damar içi (IV) şant hazırlanması ve . Ven tuzlu enjekte açık olduğundan emin olun. Not: FITC dekstranın bir IV uygulama (yüksek molekül ağırlığı, 100 ul 10 mg / ml, 2,000 kDa) FCFM femoral mikrovaskuleterini görselleştirme kolaylaştırır. Doksorubisin (100 ul, 8 mg / kg, Adriyamisin) veya tuzlu su, aynı zamanda, daha sonra önceden ısıtılmış bir kuyruk venine IV tatbik edilecektir. </li><li> Bir polistiren sahnede supinely fareyi yerleştirin. Pad fare Güvenli ve cerrahi koli bandı kullanarak konumunu korumak. </li></ol>sırasında ve Doksorubisin veya Salin İdaresi sonra FCFM tarafından Femoral Damar başlığı &quot;&gt; 3. Görüntüleme NOT: (1) mikroprob (mini0 / 30): Bu çalışmada kullanılan lifli konfokal mikroskop iki üniteden oluşmaktadır. (2) Lazer tarama ünitesi (LSU-488; 488 nm dalga boyu). <ol><li> LSU 488 nm dalga boyunda lazer kullanarak analizler her zaman tur gerçekleştirin. NOT: Ana ünite dedektörü, filtre tespit (500-650 nm) yayılan floresan. edinilen görüntüler daha sonra yeniden ve / sn 12 kare hızında görüntülenir. </li><li> Dikkatle iğneden şırınga ayırın ve FITC dekstran içeren yeni bir şırınga takın. FITC dekstran (IV) 100 ul yönetme. </li><li> Karşılık gelen bir görüntü elde etmek için z ekseni uyum sonra, bakış uygun bir alanına mikroprob (mini0 / 30), Shift ve sabitleştirmek. Açık ve odaklanmış sinyal visi- kadar solmaya ilk sinyal bekleyinble. </li><li> Kısa bir süre stabilizasyon (~ 30 sn) için bir temel kan akışını kaydedin. Sonra doksorubisin veya tuzlu ya içeren, iğne başka şırınga bağlayın. IV, 100 ul doksorubisin veya tuzlu yönetmek. </li><li> 20 dakika boyunca sürekli olarak enjekte FITC dekstran akışını izleyin. FCFM ilişkili yazılım üzerinde, kan damarlarını ölçmek için üst cetvel üzerinde çapı düğmesini kullanın ve kategorize onları küçük (&lt;15 mikron) veya büyük (&gt; 15 mm). </li><li> Anestezi doz aşımı ile hayvan Euthanize. </li></ol>

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Kemoterapi ile indüklenen vasküler toksisitesinin değerlendirilmesi nedeniyle, gerçek zamanlı olarak bir uyarıcıya tepki olarak damar dinamiklerinin görüntülenmesi güçlüğü nedeniyle zordur. Birçok klinik çalışmalar birkaç kemoterapiler doğrudan vasküler yaralanmaya neden olduğunu ima var, ama bu toksisite mekanizması ve özellikleri tam olarak açıklığa kavuşmuş. Bu tarifnamede, 8-10 tarif edildiği gibi lifli konfokal floresan mikroskobu içeren farelerde kemoterapi potansiyel vasküler toksisitesinin değerlendirilmesi için in vivo moleküler görüntüleme platformu, gerçek zamanlı kurduk. Farelerin Bu yüksek çözünürlüklü moleküler görüntüleme arteriyel kan akımını ve gemilerin mimarisi görselleştirmek için uygundur. Bu uzun bir süre boyunca aynı hayvanda tedavi kaynaklı komplikasyonlar, gerçek zamanlı belirlenmesini sağlar. Doku yanı sıra bilinen doksorubisin, in vitro olarak endotelyal hücrelere karşı toksik olduğu: Bu kemoterapi iki sınıfını değerlendirildiHerhangi bir vasküler etki eski kanıtlar çok sınırlı olan bir kontrol kemoterapi gibi doksorubisin 10-15 ve paklitaksel ile muamele edilmiş hayvanlardan elde edilen s. FCFM lazer tarama konfokal teknolojisi gerçek zamanlı floresan boyalı derin dokuları izleme ve in vivo 9 kan damarlarının time-lapse video görüntüleri üretir kolaylaştırır. Çalışmamızda FCFM bir prototip vasculotoxic madde olarak doksorubisin akut vasküler etkisi gözlemlemek için kullanılmıştır. Kısa bir süre doksorubisin uygulamasından sonra başlayan bu etki, kan damarları boyutu bağımlı oldu: küçük gemilerin çapı, daha belirgin hasar. Küçük çaplı floresans sinyali (&lt;15 um) gemi doksorubisine bağlı sabit gemi sıkışmasının bir sonucu olarak aşama aşama azalır. Hiçbir belirgin iyileşme gerçek zamanlı görüntüleme sonraki 8 dakika boyunca tespit edildi. Büyük çaplı (&gt; 15 mikron) gemiler daha az hasarlı olduğu; bütünlükkendi duvarın düzensiz yüzey sergileyen, tatlıya. Bu etkiler doksorubisin benzersiz ve bu metodoloji titizlikle ilacın spesifik etkisinin delineates belirten, paklitaksel kullanılmıştır zaman belirgindi. Değişiklikler ve sorun giderme Protokol boyunca, lazer güç damarları göstermek için başlangıç ​​kaydı sırasında değiştirilebilir. Ancak, lazer güç deney sırasında değiştirilmemelidir. Buna ek olarak, yüksek bir lazer güç süre boyunca floresans madde ağartma olabilir. Kayıt tamamlandıktan sonra Ayrıca, bu filmin parlaklığını değiştirmek ve uzunluğunu ve hızını düzenlemek mümkündür. Gemilerin Ölçümler daha sonra yapılabilir ve meydana gelen değişikliklerin takip edilebilir. Teknik ve kritik adımlar Sınırlamalar FCMF cihazı birkaç sınırlaması vardır. Faiz (ROI) bölgesi içinde değişebilirGörüntüleme zamanı. Ayrıca, görüntülü alan kurumasına olabilir; bir sulu alan tutmalı. floresan ajan çamaşır suyu olabilir ve sinyal kaybolur. Bir dikkat etmelidir bir kritik adım hayvan ve iyi ROI değişiklikleri önlemek için sabit sonda tutmaktır. Önemi ve Gelecekteki uygulamalar kurulmuş deney platformu kemoterapi ya da alternatif olarak, potansiyel vasküler toksisite profilini karakterize etmek için bir muhtemel biyolojik marker olarak yanıt için acil bir test olarak hizmet edebilir. Damar bozukluğu çalışılan mekanizması bağlı olarak, yöntem, aynı zamanda, potansiyel maddelerin değerlendirilmesinde gelecekte yararlı kemoterapi ile indüklenen vasküler toksisitesini azaltmak için belirlenebilir. Kanser kurtulan potansiyel uzun vadeli vasküler komplikasyonları azaltmak için ihtiyaç kemoterapi kaynaklı damar toksisite arkasındaki mekanizmayı keşfetmek için bize sürücüler.

Klinik kanıtlar kemoterapi çeşitli sınıfları Raynaud fenomeni, hipertansiyon, myocardialinfarction, serebrovasküler atak ve hepatik veno-occlusivedisease 1,2 ile kendini gösteren vasküler patolojilerin çeşitli ortaya belirtir. Başlangıçta bu amaçla 3-5 için geliştirilmiş, ancak az uygulanmasıyla tümör hücrelerini yok etmek için tasarlanmış olup, ancak &quot;&#39;kazara anti-anjiyojenik ilaçlar&quot;, &quot;yan mümkün anjiyojenez inhibitörleri olarak etki geleneksel kemoterapötik maddeleri tarif oldukça yeni bir terimdir mümkün 3 gibi normal hücrelere zarar &quot;. Serum biyomarkırları kullanarak klinik çalışmalarda görüldüğü gibi çeşitli kemoterapiler vasculo-toksik maddeler olarak ima edilmiştir. Bunlar arasında maddeler (örneğin, siklofosfamid gibi), platin (örneğin sisplatin gibi) bileşikleri ve antrasiklinler 1,2,5-7 alkile edilir. Akut kardiyovasküler komplikasyonlar Bir resul olarak ortaya çıkabilirKemoterapi ile uyarılan vasküler toksisite t. Onlar geç vasküler morbidite riski ateroskleroz ve hesabına gibi kronik koşulları dikkate ilerleyebilir. Ancak, klinik kanıt montaj rağmen, damar toksisite mekanizması vurgulayan belirlenen çalışmalar yetersizdir olduğunu ve bu nedenle, onlar indirebilmek tam patogenezi daha aydınlatılması garanti edilir. Kemoterapi kaynaklı vasküler toksisite mekanizması açığa büyük bir meydan okuma in vivo vasküler fonksiyonu soruşturma karmaşıklığı kaynaklanmaktadır. Biz burada kan akışını ve gemilerin özelliklerini yakalamak sağlayan farelerde in vivo moleküler görüntüleme yüksek çözünürlüklü bir platform açıklar. Bu platform, doğrudan tedaviye bağlı vasküler etkilerinin tespitini kolaylaştırır: gerçek zamanlı olarak, hem de aynı bireylerin içinde bir süre boyunca onları takip gibi.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:579px;">Name of Material/ Equipment</td>
			<td style="width:423px;">Company</td>
			<td style="width:213px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">general anesthesia (100mg/kg ketaset and 6mg/kg XYL-M2).&#160;</td>
			<td>Fort Dodge Animal Health, IA, USA and Biove Laboratories, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">a depilatory cream (Veet)</td>
			<td>&#160;ReckittBenckiser, Bristol, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A 30-gauge, 1/2-inch needle attached to 1 ml syringe&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC dextran at a volume of 100 &#181;l (10 mg/ml; FD2000S, MW 2000000 Dalton)&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxorubicin (8 mg/kg, Adriamycin)</td>
			<td>Teva, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paclitaxel (1.2 mg/kg, Medexel)&#160;</td>
			<td>Taro, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td></td>
			<td></td>
			<td></td>
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chemotherapy-induced vascular toxicity - real-time in vivo imaging of vessel impairment

Kemoterapiler bazı sınıfları vasküler morbidite riski hastayı zemin hazırlayabilir uzun vadeli koşulları içine ilerleyebilir akut vasküler değişiklikleri uygulamak olabilir. Ancak, montaj klinik kanıt olsa, vasküler toksisite ve bu nedenle damar / kardiyovasküler bozukluklar heterojen bir grup etiyolojisinde açık çalışmalar yetersizdir aydınlatılamamıştır gerekmektedir yoktur. Ayrıca, damar toksisite yatan mekanizma olabilir tamamen miyosit yaralanma doğrudan ilgilidir kemoterapi kaynaklı kardiyotoksisitenin, ilkelerinden farklı olabilir. Biz anti-kanser tedavilerinin olası akut vasküler toksisite değerlendirmek için in vivo moleküler görüntüleme platformu, gerçek zamanlı kurduk. Biz uygun kapalı organ ve referans blo içinde damarsal görselleştirmek için in vivo, farelerde yüksek çözünürlüklü moleküler görüntüleme, bir platformu kurdunuzHer birey ise aynı bireyler içinde od gemiler kendi kontrolü olarak hizmet vermektedir. Kan damarı duvarları uç organ hasarı erken bir olay olabilir vasküler toksisite benzersiz bir mekanizma temsil, doksorubisin uygulamadan sonra bozulmaktadır. Burada, lifli konfokal floresan mikroskobu (FCFM) göre görüntüleme yöntemi, hayvan deneklerde hücresel ve alt-hücresel düzeyde fizyolojik olayları anlamak için yenilikçi bir mod sağlayan, tarif edilmektedir.

Gerçek zamanlı olarak in vivo sürekli görüntülemede Burada kullanılan görüntüleme aparatı, damar görselleştirme ve kemoterapi gibi çeşitli uyarıcılara verdiği cevap sağlayan bir sonda ile donatılmış bir yüksek çözünürlüklü, lifli konfokal mikroskop vardır. Derin damarlar ya da organ görüntüleme kolaylaştırabilir rağmen, sonda için küçük bir insizyon, bu yöntem minimal invaziv. prob demetleri onlarca lifleri, mikroskop optik ve hassas özel konnektörü binlerce oluşur. Şematik bir temsili, Şekil 1 &#39;de gösterilmiştir. Femoral mikrovasküler Görüntüleme Bu damarların çap göre femur kan damar ağını sınıflandırmış (küçük &lt;15 um, büyük&gt; 15 um) FCFM ile FITC-dekstran enjekte edilmiş farelerde. Hızlı bir vazokonstriksiyon - küçük damarların (2 5 dk) doksorubisin ile oluşturuldu. Tam bir disappearanFITC dekstran floresan ce, 8 dakika sonrası doksorubisin tedavisi (Şekil 2A örneğin, oklar, Video 1). Birkaç farelerde, kan damarı floresan sinyalinin azaltılması ve onun perivasküler bölge, doksorubisin uygulama birkaç saniye sonra (Şekil 2B g, oklar) gözlenmiştir çevreleyen sinyali arttırır. Bu sonuçlar, çevre dokulara kan kabından yüksek molekül ağırlıklı dekstran bu damar geçirgenliğinin artması ve sızıntıyı göstermektedir. Resim floresans sinyali FITC-dekstran ile daha önce enjekte değil, farelerde doksorubisinin uygulanmasından sonra açıktı. Paklitaksel ile tedavi edilmiş fareler içindeki kan damarları yapısı gösterdi ve ölçüm süresi boyunca, serum fizyolojik enjekte edilen fareler (gösterilmemiş olan) gözlenenler ile akış hızı. <img alt="Şekil 1," src="/files/ftp_upload/51650/51650fig1highres.jpg" width="300" /><br/&gt; Şekil 1. FCFM iki üniteden oluşmaktadır burada kullanılan konfokal mikroskop: mikroprop (mini0 / 30) ve lazer tarama birimi (LSU-488; 488 nm dalga boyu).. <img alt="Şekil 2," src="/files/ftp_upload/51650/51650fig2highres.jpg" /> Şekil 2. Femoral mikrovasküler FITC dekstran floresan sinyal görüntüleri. Gemiler dichotomously (&lt;15 mikron ya da majör minör) sınıflandırıldı (&gt; 15 mikron) kendi kalibre göre. FITC dekstran femoral mikrovasküler (100 ul 10 mg / ml) eklenmiştir. (A) FCFM tarafından görüntüsü küçük kaplar, fareler önce ve doksorubisin ya da tuz ya da IV uygulaması sırasında görüntülenmiştir enjekte 2 dakika doksorubisin uygulamadan sonra akut vazokonstriktör başladı (f , n esnasında sinyal hiçbir kurtarma belirgin iyileşme ile, tamamen ortadan kalkması kadar floresan sinyal sürekli daralması gösteren) arrowext gerçek zamanlı görüntüleme (g ok) sekiz dakika. (B) Görünüm, bazı anlık olarak, enjeksiyondan hemen sonra kemoterapi-enjekte edilen farelerin (g ok) kan damarları duvarları etrafında bir &quot;puslu&quot; alan, potansiyel gösterir dekstran-FITC kaçağı. <a href="https://www.jove.com/files/ftp_upload/51650/51650fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Video 1 Femoral mikrovasküler görüntüleme. Bir temsilci FITC dekstran floresan minör (çapı &lt;15 mikron), kan damarları filmini fotoğrafladı. 100 ul FITC-dekstran (10 mg / ml) ile enjekte edilmiş fareler görüntülenmiş ve doksorubisin IV uygulamasına andan itibaren fotoğraflandı: Yumurtalık ve femoral mikrovasküler zaman fotoğraflar ve yalar. 2 - doksorubisin, enjeksiyondan sonra 5 dakika, küçük damarları floresan sinyalinin tamamen kaldırılması, Terbiyede sonra daha önce 8 dakika süre ile dramatik bir vazokonstriksiyona gösterdit. (AVI) <a href="https://www.jove.com/files/ftp_upload/51650/video1.mpeg" target="_blank">Bu videoyu görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment at JoVE.com

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Kemoterapi kaynaklı Vasküler Toksisite - Gerçek zamanlı<em&gt; In vivo</em&gt; Gemi Düşüklüğü Görüntüleme

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

chemotherapy-induced-vascular-toxicity-real-time-vivo-imaging-vessel

Nanyang Technological University

Research

JoVE Journal

Medicine

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

7.3K Görüntüleme.  Tel Aviv University. We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Video: Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Jinekolojik Onkoloji Sentinel lenf nodu intraoperatif Algılama için Multispektral Gerçek zamanlı Floresans Görüntüleme

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

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Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Kontrast Geliştirilmiş Gemi Görüntüleme MicroCT kullanarak

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital Görüntüleme Gerçek Zamanlı tümörler Nanopartikül Alımı Değerlendirme

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

An<em&gt; In vivo</emKan Gemi Geçirgenlik Test&gt; Test

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Boyuna, Non-invaziv için Eye Ön Kamara içine Nakli<em&gt; In vivo</emGerçek zamanlı Tek hücreli Çözünürlük&gt; Görüntüleme

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

Yenidoğan Fare Akciğer Akciğer Akışkan Ciltler Gerçek zamanlı X-ışını görüntüleme

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Konfokal Optik Görüntüleme ile Rose Bengal Fototromboz<em&gt; In Vivo</em&gt;: Tek Gemi İnme Bir Model

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Ferric Klorür kaynaklı Karotis Arter ve Mezenter Gemi Tromboz Fare Modeli

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Tümör mikroçevresinde Gerçek zamanlı Multisellüler Dynamics Genişletilmiş Time-lapse intravital Görüntüleme

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Akut miyokard infarktüsünde farelerde gerçek zamanlı basınç-hacim Analizi

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Kemoterapi kaynaklı Vasküler Toksisite - Gerçek zamanlı

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