Bu çalışmada CCSRI Hibe # 700.537 ve CIHR Hibe JDL ve NIH / NCI hibe # CA120711-01A1 ve AZ CA120711-01A1 # 84.535 tarafından desteklenmiştir. Tüm deneyler Kurumsal Hayvan Bakım yönetmelik ve yönergeler doğrultusunda gerçekleştirilen ve California San Diego, Hayvan Bakım Üniversitesi&#39;nde Komitesi kullanın ve Western Ontario Üniversitesi&#39;nde kullanın.

1. Kuş embriyo CAM içine tümör Aşılama <ol><li> Kabuk az döllenmiş tavuk embriyolar 8 açıklandığı gibi hazırlayın. </li><li> Embriyonik gelişim Gün 9, 1 ml şırınga üzerine bağlı 18-gauge iğne kullanılarak bir mikro enjektör montajı. 5-6 inç Tygon boru (1 / 32 &quot;iç çapı, 3 / 32&quot; dış çapı, 1 / 32 &quot;duvar kalınlığı) bir parça kesin ve tüp içine iğne konik dikkatlice yerleştirin. Tüp yaklaşık 4-5 cm iğne ucu kadar uzanır (Şekil 1a). </li><li> Hücre süspansiyonu ile şırınga ve tüp doldurun. Daha sonra, boru sonunda mikroenjeksiyon cam iğne takın ve dikkatli bir şekilde herhangi bir hava kabarcıkları. </li><li> Gün ışığı ile CAM (Şekil 1c) içinde bolus olarak 10,000-100,000 kanser hücreleri bir diseksiyon kapsamında 9 embriyolar (Şekil 1b) enjekte edilir. Hücreleri enjekte yavaş yavaş ve dikkatli bir iğne CAM içinde görünür bir bolus oluşturmak için hücreleri için doğru yerde olduğundan emin olun. Hücreler CAM yüzeye damla Kimwipe veya diğer aplikatör kullanılarak temizlenebilir. </li><li> % 60 nem ve 38 ° C&#39;de nemlendirilmiş inkübatör embriyo dönün ve tümör izin büyümeye ve vascularize (7 güne kadar). </li></ol> 2. Nanoparçacıkların hazırlanması <ol><li> Tavuk embriyo içine enjeksiyon için floresan etiketli CPMV nanopartiküller hazırlamak için; viral nanopartiküller (herhangi kaldırmak için kullanarak ve bir dakika boyunca santrifüj önce 100 mg / ml Vortex karışımı bir konsantrasyon, PBS, pH 7.4 8 olarak sentezlenmiş sulandırmak toplar. Sonikasyon konjugatları ve agregasyon derecesi bağlı olarak yararlı olabilir 4 saklandığında floresan etiketli CPMVs stokları en az 1 yıl boyunca stabildir ° C karanlıkta bir çift koyarak stokları periyodik olarak kontrol edin bir cam slayt üzerine düşer ve floresan kontrol Transmisyon Elektron Mikroskobu (TEM) parçacıklar konjugasyon sonra değişmediğini belirlemek için kullanılır. </li><li> Kısaca, viral nanoparçacıkların bir mikrogram (2 ul nihai hacmi) bakır kaplı ızgaraları eklenebilir. Sonra, bir eşit miktarda elektron mikroskobu (Philips EM 410) az 1 dakika süreyle% 2 oranında uranil asetat ekleyin. </li></ol> 3. Floresan etiketli viral nanoparçacıkların İntravenöz enjeksiyon <ol><li> 16 Gün, yukarıda açıklandığı gibi mikro enjektör montajı. Şırıngaya boru (&gt; 200 mcL) ile viral nanoparçacık istenilen hacmi çizin. Herhangi bir hava kabarcığı dikkatlice çıkarın. Mikroenjeksiyon cam boru sonunda iğne takın. Iğne kadar uzun ve mümkün olduğunca konik (Şekil 2a) olduğundan emin olun. Iğne çok künt ise, ektoderm ile delmek için mümkün olmayacaktır ve gemi nüfuz başarısız olur. Iğne çok keskin ise doku nüfuz ederken, ucu çökecek. </li><li> İstediğiniz siteye distal tümör taşıyan embriyo haline intravenöz (Şekil 3d) görüntülenebilmekte 1 mg / ml flöresein dekstran (MW 70.000 Da, Invitrogen) 100 ul enjekte edilir. Başarılı CAM ven kanülasyonu enjeksiyon akış yolu kan temizleme (Şekil 2b) tarafından açıktır. Enjeksiyon sonrası vasküler sızıntı 1 saat değerlendirin. </li><li> İstediğiniz siteye distal vasküler sızıntı tümörleri içeren embriyolar halinde intravenöz CPMV-Alexa Fluor 647 (CPMV-AF 647) veya pegile CPMV-Alexa Fluor 647 (CPMV-PEG-AF 647) 1 mg / ml solüsyon enjekte 50 ul görüntülenebilmekte. </li><li> Görüntü hemen tümörler ve enjeksiyondan sonra bir Yokogawa dönen disk, Hamamatsu ImagEM 9100-12 EM-CCD kamera ile donatılmış bir Zeiss Examiner Z1 dik bir mikroskop kullanılarak her saat. </li></ol> 4. Gerçek zamanlı intravital görüntüleme <ol><li> Embriyo görüntüleme birimi bir araya getirmek için, vakum gres embriyo görüntüleme ünitesi kapağın alt görüntüleme liman çevresinde ince bir tabaka halinde uygulayın ve liman üzerine 18 mm cam lamel uyacak. </li><li> Lamel görüntüleme için istediğiniz alanı kapsayacak böyle embriyo yerleştirin. Lamel yavaşça embriyo ile temas edinceye kadar kapağı düşük ve daha sonra birim üzerine kapağı vidalı bir yerde tutun (Şekil 2c). </li><li> Embriyo içeren çanak dışında embriyo görüntüleme ünitesi içine 37 ° C sıcak su ekleyin ve sonra 37 dengelenmiş içinde çevre odasına ° C ile dik bir konfokal mikroskop sahneye tüm birim </li><li> İplik disk konfokal floresan mikroskobu (Şekil 2e) altında embriyo içeren Pozisyon görüntüleme ünitesi. Görüntüleme ünitesi yakalanan üç boyutlu Z yığınları ve zaman atlamalı görüntüleri için izin, yer embriyo tutun ve görüş alanı yakalama görüntüleri ise sabit tutmak. Biz kazanmak ve Perkin Elmer (eski Doğaçlama) Volocity yazılım paketi (Şekil 3a) kullanarak üç boyutlu zaman atlamalı görüntüleri analiz. Tümörün üç boyutlu bir yığın yüksek çözünürlüklü ve detaile görselleştirmek için çevreleyen damar kazanmakd belirli bir zaman noktalarında yapısal analizler. Tümör damarsal ayrıntılı yapısal değişiklikler eşlemek için normal süre noktaları üç boyutlu yığınlar edinin. Görüntü tümörler her enjeksiyondan sonra saat. </li><li> Hesaplayarak ortalama Alexa Fluor (AF), tümör ya da böyle bir Volocity olarak stroma (non-tümör alan) kullanarak görüntü kantitatif yazılımı (Perkin Elmer) seçilen bölgeler içinde 647 sinyal alımını viral nanoparçacıkların Asitli. Stroma oranı tümör stroma ortalama AF 647 sinyal üzerinde tümörün ortalama AF 647 sinyal bölünmesi ile hesaplandı. Tümör / stroma oranı 1&#39;den yüksek bir nanoparçacık tümörün alındığını olduğunu gösterir. </li></ol> 5. Temsilcisi sonuçları: Örneğin burada anlatılan, biz günde 9 tavuk embriyolar (Şekil 1b) CAM içinde yaklaşık 1 mm boyutunda bir bolus oluşturmak için HT-29 kolon kanseri hücreleri enjekte etti. Aşılama sonra, embriyolar yeterli tümör büyümesi ve vaskülarizasyon (Şekil 1c) izin nemlendirilmiş bir inkübatör 7 gün süreyle kültüre edildi. Embriyolar tümör vaskülarizasyon onaylamak düşük molekül ağırlıklı dekstran ile intravenöz enjekte edildi ve tümörler Zeiss AxioExaminer Z1 dik mikroskobu (Şekil 2d) altında görüntülendi. CPMV-AF 647 veya CPMV-PEG-AF 647 nanopartiküller intravenöz uygulamadan sonra (Şekil 3a ve b), yüksek çözünürlükte gerçek zamanlı konfokal görüntüleme (Şekil 2e) CPMV ve CPMV-PEG hızla tüm damarsal etiketli nanopartiküller saptandı, ancak tümör CPMV-PEG alımı (Şekil 3a) 12 saat sonra, CPMV daha yaklaşık 3 kat daha yüksek oldu. Nanoparçacıkların nispi tümör alımı, görüntü analiz yazılımı (Perkin Elmer Volocity) kullanılarak tespit edildi. Tümör içinde ve dışında ilgi Bölgeler seçildi (stromal bölmesi) ve her birinin ortalama floresan yoğunluğu tespit edilmiştir. Veri tümör / stroma oranı olarak ifade edilir. <img src="/files/ftp_upload/2808/2808fig1.jpg" alt="Şekil 1"/> Şekil 1 bir kuş embriyo CAM içine tümörlerin Mikroenjeksiyon. Belirtildiği gibi (a) mikroenjeksiyon cihaz bileşenleri bir araya. (B) 9 gün Kuş embriyolar CAM tüm yüzeyini kapsayacak şekilde yayıldı tümör ile aşılanmış olmak için hazırız. (C) 16 gün, tümör çapı 1 cm (kesikli çizgi) kadar büyüdü ve nanopartiküller ile enjeksiyon için hazır olacak. <img src="/files/ftp_upload/2808/2808fig2.jpg" alt="Şekil 2"/> Şekil 2 Nanopartikül enjeksiyon ve intravital görüntüleme. Gösteren bir diseksiyon mikroskop altında enjeksiyon (a), (temizleyerek görülen CAM damar içine enjekte edilir ve (b) mikroenjektör iğne kan akımı enjekte nanopartiküller damar içine eklenir (ok ile gösterilir) ve hazır mikroenjektör iğne ucu kan). (C) Görüntüleme birimi lamel CAM ile doğrudan arabirim ile kuş embriyo içeren. (D) nanoparçacık enjeksiyon öncesinde, tümör vaskülarizasyon floresein dekstran enjeksiyondan sonra intravital görüntüleme ile değerlendirildi. (E) Görüntüleme Ünitesi, 37 ° C sıcaklığa ayarlı muhafaza kümesi içinde dik bir konfokal mikroskop sahnede yer embriyo içeren <img src="/files/ftp_upload/2808/2808fig3.jpg" alt="Şekil 3"/> Şekil 3 insan tümörlerinde nanoparçacık alımı intravital görselleştirme. Tümörler (a) CPMV-AF647, (b) CPMV-PEG-AF647 enjeksiyonundan 7 saat sonra görüntülenir. d) sonraki analiz için kuş embriyo tümör eksizyonu.

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Kuş embriyo chorioallantoic membran (CAM), vasküler dinamikler ve insan tümörlerinde farmakokinetiği değerlendirmek için kullanışlı bir modeldir. CAM yapısı ve konumu, yüksek kalitede görüntü elde etme sağlar ve invaziv cerrahi prosedürler olmaksızın birçok türde kanser xenografts barındırmaktadır. Ayrıca, kanser tümörü xenografts, tümör dokusu nanoparçacıkların birikimi değerlendirmek için hızlı, ucuz ve yarı-yüksek verimlilik anlamına gelir sunan, 7 gün içinde vaskülarize haline chorioallantoic membran yerleştirilir. CAM kabuk az tavuk embriyoların implante kanser xenografts tümör damarsal nanoparçacık alımı ile ilgili bir dik Epifloresans veya konfokal mikroskop, bağlamsal ve zamansal bilgiler yüksek çözünürlüklü optik erişilebilir olduğundan kolayca elde edilebilir. Bu modelde Kanser xenografts az 200 m derinlikte kalarak büyük tümörlerde, CAM üzerinde yanal büyümek eğilimindedir. Standart Epifloresans mikroskopları tüm tümör kitlesi etkin bir şekilde nüfuz eder, çünkü bu, onları özellikle iyi intravital görüntüleme için uygun hale getirir. Buna karşılık, fare içinde yüzeyel veya ortotopik siteleri ya implante tümörler zor non-invaziv teknikleri ile bu tümörler içinde derin nanopartiküller doğru lokalize etmek, üç boyutlu olarak çoğalırlar. Biz geniş bir yelpazede nanoparçacık formülasyonların in vivo analizi için uygun olduğu için bu model için potansiyel vurgulayarak, kuantum noktaları, lipozomlar ve demir oksit, insan tümör xenografts bir dizi nanopartiküller alımını değerlendirmek için bu model kullanılır.

<table border="1"><tbody><tr><th> Reaktif Adı / Ekipman </th><th> Şirket </th><th> Katalog Numarası </th><th> Yorumlar </th></tr><tr><td> Döllenmiş leghorn yumurta </td><td> Frey `ın Kuluçkahane, Jacobs </td><td> N / A </td><td></td></tr><tr><td> Dremel döner aracı </td><td> Dremel </td><td></td><td> Herhangi bir model kullanılabilir </td></tr><tr><td> Hiçbir Dremel kesme tekerlekleri. 36 </td><td> Dremel </td><td> 409 </td><td></td></tr><tr><td> Sporcu çıkım </td><td> Berry Hill </td><td> 1550HA </td><td></td></tr><tr><td> Sporcu inkübatör </td><td> Berry Hill </td><td> 1502EA </td><td></td></tr><tr><td> Etanol </td><td></td><td></td><td> % 70 (hacim / hacim) </td></tr><tr><td> Polistiren tekneler tartmak </td><td> VWR </td><td> 12577-01 </td><td></td></tr><tr><td> Kare Petri kutularına </td><td> Simport, VWR </td><td> 25378-115 </td><td></td></tr><tr><td> Rubbermaid kauçuk konteyner kapaklı </td><td> Guillevin </td><td> RH3-228-00-BLU </td><td> delik tarafı içine delinmiş </td></tr><tr><td> Dikey pipet çektirmesi </td><td> David Kopf Aletleri </td><td> modeli 720 </td><td> Ayarlar: 16.3 (ısıtıcı) ve 2.3 (solenoid) </td></tr><tr><td> Sodyum borosilikat camdan kılcal tüpler </td><td> Sutter Enstrüman </td><td> BF100-58-10 </td><td> (OD, 1.0 mm; ID 0.58 mm, 10 cm uzunluk) </td></tr><tr><td> 1X Dulbecco&#39;nun Modifiye Kartalı Medium (DMEM) </td><td> Invitrogen </td><td> 11995073 </td><td></td></tr><tr><td> Dulbecco&#39;nun </td><td> Invitrogen </td><td> 14190250 </td><td> pH: 7.4 </td></tr><tr><td> Fosfat Tamponlu Salin (D-PBS) (1X), sıvı </td><td></td><td></td><td></td></tr><tr><td> Tripsin, EDTA 4NA% 0,05 (1X), sıvı </td><td> Invitrogen </td><td> 25300054 </td><td></td></tr><tr><td> Fetal sığır serumu </td><td> Invitrogen </td><td> 12483-020 </td><td> Inaktive Isı </td></tr><tr><td> Hemasitometre </td><td> Hausser Bilimsel, VWR </td><td> 15170-090 </td><td></td></tr><tr><td> Santrifüj </td><td> Eppendorf modeli 5810R </td><td> 5811 000.010 </td><td></td></tr><tr><td> İnce nokta forseps </td><td> VWR </td><td> 25607-856 </td><td></td></tr><tr><td> Tygon R-3603 tüp </td><td> VWR </td><td> 63009-983 </td><td> 50 ft (1/32-inch iç çapı, dış çap, 3/32-inch 1/32-inch duvar kalınlığı </td></tr><tr><td> Enjeksiyonları 18-gauge iğne için derialtı iğneler </td><td> BD </td><td> 305195 </td><td> 100 Kutu </td></tr><tr><td> Enjeksiyon için 1 ml şırınga </td><td> BD </td><td> 309602 </td><td> 100 Kutu </td></tr><tr><td> Fiber-optik mikroskop aydınlatıcı </td><td> Amscope </td><td> HL250-AY </td><td> 150W </td></tr><tr><td> Kimwipes </td><td> VWR </td><td> 10805-905 </td><td></td></tr><tr><td> V. unguiculata tohumları (California siyah-gözü 5) </td><td> Burpee </td><td> 51771A </td><td></td></tr><tr><td> Kapalı büyüme ışıkları </td><td> Sunlite, Gardener&#39;ın Kaynağı </td><td></td><td></td></tr><tr><td> Metil-PEO4 NHS ester </td><td> Delmek </td><td> PI22341 </td><td></td></tr><tr><td> MPEG-NHS, PEG succinimidyl ester, MW 2000 </td><td> NANOCS </td><td> PEG1-0002 </td><td></td></tr><tr><td> Alexa Fluor 647 karboksilik asit (succinimidyl ester) </td><td> Invitrogen </td><td> A20006 </td><td></td></tr><tr><td> Oregon Yeşil 488 succinimidyl ester * 6-izomeri * </td><td> Invitrogen </td><td> O-6149 </td><td></td></tr><tr><td> Dimetil sülfoksit (DMSO) </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Dibazik monohydrogen fosfat </td><td> Sigma </td><td> 379980 </td><td> KH2PO4 (phopshate tampon) </td></tr><tr><td> Mono dihidrojen fosfat </td><td></td><td> P5655 </td><td> KH2PO4 (phopshate tampon) </td></tr><tr><td> Superose 6 boyut-dışlanma sütun </td><td> GE sağlık yaşam bilimleri </td><td> 17-0673-01 </td><td></td></tr><tr><td> Akta Explorer 100 Kromatografi </td><td> GE Amersham Pharmacia </td><td> WS-AKTA100 </td><td></td></tr><tr><td> Akta yüksek akış kiti </td><td> GE sağlık yaşam bilimleri </td><td> 18-1154-85 </td><td></td></tr><tr><td> Sakaroz </td><td> Sigma </td><td> S0389 </td><td></td></tr><tr><td> Ultrasantrifüjdeki </td&gt; <td> Beckman </td><td></td><td></td></tr><tr><td> GB 28 Ti rotor </td><td> Beckman </td><td> 342204 </td><td> Salıncak kova </td></tr><tr><td> 50.2 Ti rotor </td><td> Beckman </td><td> 337901 </td><td> Sabit açılı </td></tr><tr><td> Amicon Ultra-15 Santrifüj Filtre Üniteleri </td><td> Millipore </td><td> UFC910008 </td><td> 100 kDa kesti </td></tr><tr><td> Gradient eski </td><td> Biorad </td><td></td><td></td></tr><tr><td> dekstran, floresein, 70.000 MW, anyonik </td><td> Invitrogen </td><td> D1823 </td><td></td></tr><tr><td> İplik disk konfokal floresan mikroskop </td><td> Nisabı; Yokogawa CSU, 10, Yokogawa </td><td> N / A </td><td></td></tr><tr><td> Epifloresans geniş alan mikroskobu </td><td> Nisabı; Zeiss Axio Examiner, Zeiss </td><td> N / A </td><td></td></tr><tr><td> Hamamatsu ImagEM 9100-12 EM-CCD kamera </td><td> Nisabı; Hamamatsu </td><td> N / A </td><td></td></tr><tr><td> Mikroskop için sıcaklık muhafaza ünitesi </td><td> Hassas Plastik </td><td> N / A </td><td></td></tr><tr><td> Vakum gres </td><td> VWR </td><td> 59344-055 </td><td></td></tr><tr><td> Dairesel cam yok lamelleri. 1 (18 mm) </td><td> VWR </td><td> 16004-300 </td><td></td></tr><tr><td> Volocity yazılım </td><td> Perkin Elmer </td><td></td><td></td></tr><tr><td> Civciv embriyo muhafaza </td><td></td><td> özel imal </td><td></td></tr><tr><td> Hassas makas </td><td> VWR </td><td> 25608-203 </td><td></td></tr><tr><td> Formalin </td><td> Bioshop </td><td> FOR201.500 </td><td> Fumehood olarak kullanın </td></tr><tr><td> Optimal Kesme </td><td> Fisher; Doku Tek </td><td> 1437365 </td><td></td></tr><tr><td> Sıcaklık (OCT) </td><td></td><td></td><td></td></tr><tr><td> Plastik kalıpları </td><td> Balıkçı </td><td> 22-038217 </td><td></td></tr><tr><td> VWR VistaVision HistoBond Yapıştırıcı Slaytlar </td><td> VWR </td><td> 16004-406 </td><td></td></tr><tr><td> DAPI ile altın Prolong </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td> Kriyostat için tek kanatlı </td><td> Balıkçı </td><td> 12-634-2 </td><td></td></tr><tr><td> Kriyostat </td><td> Leica CM 3050 S </td><td> 14047033518 </td><td></td></tr></table>

evaluation of nanoparticle uptake in tumors in real time using intravital imaging

Mevcut teknolojiler gibi ultrason, MR, PET ve CT gibi tümör görüntüleme için uygun bir xenograftlarının modeli yararını vurgulayarak, mikroskobik düzeyde 1,2,3 tümörleri nanoparçacık alımını değerlendirilmesi için yüksek çözünürlüklü görüntüler veremezken Ayrıntılı alımı analizleri yapmak için hangi. Burada, değiştirilmiş bir kabuk, tavuk embriyo modeli insan tümör xenografts nanoparçacık alımını değerlendirmek için, yüksek çözünürlüklü intravital görüntüleme kullanın. Tavuk embriyo modeli özellikle in vivo analizlerde, insan tümörlerinin büyümesini destekleyen, nispeten ucuz ve 4,5 anesthetization veya cerrahi gerektirmez, çünkü bunlar için iyi uygundur . Chorioallantoic membran (CAM) 6 içine implante Tümör hücreleri, 7 gün içinde tamamen vaskülare xenografts oluşturur . Ortaya çıkan tümörler, ya ev sahibi veya tümör sistemleri üzerinde çok az etkisi ile 72 saate kadar korunabilir non-invaziv, gerçek zamanlı, yüksek çözünürlüklü görüntüleme tarafından görüntülenmiştir. Tümör distal uygulanan boyutları ve formülasyonları geniş bir yelpazesi ile Nanopartiküller görüntülenebilmekte ve kan yoluyla akışı gibi sayısal olabilir, sızdıran tümör damarsal extravasate ve tümör yerinde biriken. Biz burada, yakın-kızılötesi floresan boyalar ve / veya polietilen glikol (PEG) polimerler 7, 8, 9,10,11 süslenmiş Börülce mozaik virüsü (CPMV) türetilen nanoparçacıkların analizi açıklanmaktadır . Intravenöz üzerine, bu viral nanopartiküller dışında damar küresel etiketleme ve tümör 7,12 içinde hızla endotel hücreleri tarafından içselleştirilmiş. Viral nanopartiküller PEGylation plazma yarılanma ömrü artırır, kan dolaşımını zamanlarını uzatır ve sonuçta gelişmiş geçirgenlik ve retansiyonu (EPR) etkisi 7, 10,11 ile tümörlerin birikimi artırır. Görüntü analiz yazılımı kullanarak bir tümör nanoparçacıkların birikimi hızı ve kapsamı zamanla ölçülür. Bu teknik, hem de görselleştirmek ve insan tümörlerinde nanoparçacık dinamiklerini ölçmek için bir yöntem sağlar.

Watch this Scientific Journal Video about Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging at JoVE.com

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

Biz bir kuş embriyo modeli dinamik, gerçek zamanlı intravital görüntüleme kullanarak insan xenografted tümörlerin vasküler nanoparçacık yerelleştirme ölçmek için yeni bir yaklaşım sunuyoruz.

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 ...

evaluation-nanoparticle-uptake-tumors-real-time-using-intravital

Nanyang Technological University

Research

JoVE Journal

Medicine

18.2K Görüntüleme.  University of Western Ontario. Biz bir kuş embriyo modeli dinamik, gerçek zamanlı intravital görüntüleme kullanarak insan xenografted tümörlerin vasküler nanoparçacık yerelleştirme ölçmek için yeni bir yaklaşım sunuyoruz.

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

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 ...

Tıp

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Kantitatif Gerçek-zamanlı PCR (qPCR) tarafından Prostat tümörlerinde mikroRNA Algılama

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Sıçan Cremaster&#39;ın kas iskemi-reperfüzyon Hasarı içinde Lökosit-endotelyal Etkileşim Gerçek zamanlı Dijital Görüntüleme (IRI)

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Hareket Performans Gerçek zamanlı geri bildirim ile yeniden eğitimi

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

Floresan SapC-DOPS Nanovesicles kullanma Beyin Tümörleri ve Artrit Vivo Optik Görüntüleme

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

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

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

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 ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

İnsan Tüm Kan gerçek zamanlı sitotoksisite deneyleri

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

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 ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Kantitatif gerçek zamanlı PCR ile fare meme tümör modellerinde akciğer metastazı Değerlendirilmesi

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

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 ...