We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

Bu çalışmada kullanılan tüm hayvanlar Oftalmik ve Vizyon Araştırma Hayvanların Kullanım ARVO Tablosuna göre bakım ve ele alınmıştır. HEK-293 hücrelerinden 17,18 süreli transfection ile Rekombinant AAV (rAAV), 1. Üretim NOT: McClure C, (2011) 19 vallahi bak. <ol><li> AAV vektörü plazmid geniş ölçekli bir plazmid hazırlama (en az 1 mg / ml) ile saflaştınlır. 3 plazmidler kullanın. AAV ters terminali olmadan çoğaltma ve kapsid genleri taşıyan AAV yardımcı plazmid ITR&#39;ler, AAV ITR transferi plazmid olarak anılacaktır taşıyan transgen ifade kaseti, adenovirüs ve yardımcı genleri E2, E4, ve VA RNA genleri tedarik plazmid sevk edilen tekrarlar pHELPER olarak. </li><li> Bu 3 plazmidler polietilenimin (ya da diğer transfeksiyon reaktifi) kullanılarak 293 hücreleri transfekte. Transfeksiyon verimi% 80 üzerinde iyi vi için gerekli olanral verimleri. </li><li> Bir iyodiksanol degrade üzerinde 293 hücre lisatlanndan rekombinant AAV arındırın. % 15,% 25,% 40,% 60 iyodiksanol gradyanı elde etmek için kullanılır. AAV parçacıkları ihtiva eden% 40 iyodiksanol fraksiyonu toplamak% 0.001 Pluronic PBS&#39;ye 1 saat karıştırıldı ve tampon değişimi (fosfat tamponlu tuzlu su) için 500,000 x g&#39;de ultrasantrifugasyon sonra fraksiyonu konsantre edilir. </li><li> Viral genomik titresi 20 belirlemek için, qPCR ardından DNAz ProteinaseK özet gerçekleştirin. ileri ve geri primerler AAV2 ITR bulunan 62 bp bölgesini yükseltmek. Plazmid standart için, prob FAM ile etiketlenmiş ve (BHQ) söndürücü bir kara delik vardır. </li><li> ITR primeri, 100 nM ileri ITR primeri, 100 nM, AAV2 ITR probu, 0.4 mM dNTP, 2 mM MgCI2, 1 x Platin Taq tampon, 1U Platin ters 340 nM kullanılarak 25 ul nihai hacim içinde qPCR (kantitatif polimeraz zincir reaksiyonu) yapınız Taq ve 5 ul şablonu (standart, numune ve hiçbir şablon kontrolü). </li><li> Içinstandart seri seyreltileri ile 10 katı 7 x transfeksiyon için plazmid kullanımı (5 ul 10 Ekim-10 Nisan genomları / ml nihai bir konsantrasyonda elde edilen, 2 x 10 9 3 10 ila 2 x genomları / ml her biri). </li><li> Denatürasyon için 40 döngü, ardından 15 dakika boyunca 95 ° C&#39;de bir ilk denatürasyon adımı programı başlayın 1 dakika tavlama / uzatma için 95 ° C &#39;de 1 dakika süre ile 60 ° C&#39;de ilave edildi. Kısa süreli depolama için veya uzun depolama süreleri boyunca -20 ° C &#39;de 4 ° C&#39; de muhafaza edin. </li></ol> AAV 2. Intravitreal <ol><li> Ketamin (50 mg / kg) ve ksilazin (10 mg / kg) ile C57BL6J fareler anestezi ve refleks, uygun bir anesthetization gösteren çekme refleksi ve kuyruk kıskaç yanıt doğrultma kaybını kontrol edin. Neosynephrine% 5 kornea uygulama ile öğrencilerin genişletmek ve göz damlası% 0.5 mydriaticum. Anestezi altında iken kuruluğu önlemek için göze veteriner merhem kullanın. </li><li> Bir ultra ince 30 G disposab geçmekle ekvator boyunca iğne ve vitreus boşluğuna, limbusa yanındaki (Chiu K, vallahi (2007) 21). Vitröz kavitesine merkezi iğne, doğrudan gözlemiyle ShH10-GFP veya rAAV5-GFP 1 ila 10 x 4 11 vp ihtiva eden 1 ul stok (Şekil 1) enjekte edilir. ILM deney için bir kontrol olarak, bir göz kullanın. BRB deneyler için her iki gözleri enjekte edilir. Enjekte gözleri bir anti-inflamatuar ve antibakteriyel topikal tedavi uygulayın. </li><li> Neosynephrine ile öğrencileri genişletmek ve mydriaticum göz görüntüleme için düşer. GFP (Yeşil Floresan Protein) ifadesi 1 hafta, 2 hafta, 1 ay, intravitreal enjeksiyondan sonra 2 ay (Şekil 1) takip göz fundus kamera ile fundus muayeneleri gerçekleştirin. CO 2 inhalasyon 1 ila 2 ay sonrası enjeksiyon yoluyla fareler Kurban. </li></ol> 3. İmmünohistokimya <ol><li> Retina cryosections (Şekil 1) için, l kaldırmak için enüklee gözleri teşrih1 saat süresince% 4 paraformaldehit içinde sapiens ve kornea ve daldırma düzeltme. <ol><li> Oda sıcaklığında 1 saat, oda sıcaklığında bir saat için% 20 sakroz,% 10 sukroz içinde daha önceden belirlenmiş gözleri cryoprotect. Gece boyunca 4 ° C&#39;de% 30 sukroz çözeltisi içerisinde cryoprotect. Freeze ve Cryo-matris olarak reçine gömme eyecups embed. 10 mikron cryosections olun ve özel dondurulmuş doku bölümleri için tedavi slaytlar üzerine monte ve boyama sırasında doku uyumu artırmak. </li><li> 5 dakika boyunca PBS pH 7.4 içinde% 0.1 Triton X100 ile bölümleri geçirgenliği. PBS Yıkama 2x ve blok,% 1 BSA ile PBS içinde oda sıcaklığında 1 saat,% 0.1 Tween 20. </li></ol></li><li> Retina flatmounts (Şekil 1) için, diseksiyon kolaylaştırmak için, 15 dakika süreyle% 4 paraformaldehid içinde enüklee gözleri düzeltin. <ol><li> 15-30 dakika içinde, kornea ve lens çıkarmak için gözleri teşrih. Ora serrata etrafında keserek retina pigment epiteli (RPE) ve sklera gelen retina ayırın veoptik sinir. Retina ve kalıt retina hücrelerinde floresan proteini (tespit 24 saat aşmamalıdır) sabitlemek için başka bir 30 dakika boyunca% 4 paraformaldehid içinde bırakın. Steril PBS, pH 7.4 içinde 5 dakika yıkanır. </li><li> (% 1 BSA,% 2 normal keçi ya da eşek serumu PBS,% 0.5 Triton X-100), bloke edici tampon ile PBS değiştirin ve oda sıcaklığında 4 saat ya da gece boyunca 4 ° C&#39;de ya da blokaj tamponunda inkübe edilir. </li></ol></li><li> Oda sıcaklığında ya da gece boyunca 4 ° C&#39;de ya da 4 saat süre ile blokaj tamponu içinde primer antikorlar ile dokuların inkübe edin. PBS ile yıkayın 3x. NOT: Aşağıdaki olarak kullanılan antikorlar: Anti-laminin (/ 1,000 1) etiketleme ILM anti-rodopsin klon 4D2 (1/500) etiketleme çubuklar, anti-glutamin sentetaz klonu GS-6 (1 / 1.500) etiketleme Müller hücreleri PNA lektin (1/40) etiketleme kozalakları. </li><li> Oda Tempe 1 saat (halinde kriyoseksiyonlar) ya da 2 saat (flatmounts) blokaj tamponunda Alexa Fluor konjuge sekonder antikor: 500 oranında seyreltilmiş 1 ile dokuları inküberature ve ışıktan korunan. PBS ile yıkayın 3x. </li><li> Retinaya giderici kesim yapmak ve fotoreseptör veya retina ganglion hücre (RGH) iki tarafında yukarı bakacak şekilde bir cam slayt üzerine flatmount. 4 ° C&#39;de lamel ve mağaza uygulamak, sulu mountingmedium ekleyin. </li><li> Lazer tarama konfokal mikroskop konfokal mikroskopi gerçekleştirin. Uyarma ve emisyon karışma azaltmak için, satır görüntüleri sırayla, çizgi edinin. Nyquist-Shannon örnekleme teoremine göre adım boyutunu tanımlayın. Son görüntülerde doygun piksel en aza indirmek pozlama ayarlarını kullanın. Z-proje fonksiyonu altında maksimum şiddeti kullanarak ve nihayet 8-bit RGB renk moduna dönüştürmek tek düzlemde FIJI ile Süreci 12-bit görüntüleri, proje Z-bölümleri. </li></ol> Fare Kan Örneklerinin 4. PCR Analizi <ol><li> Anestezi C57BL6J farelerin penil ven içine bir AAV-GFP 1 10 4 x 11 partikülleri içeren 100 ul stok (izofluran% 5 enjekteultra-ince 6 mm&#39;lik bir iğne ile bir insülin şırıngası kullanılarak yakın bir bölmesi içinde indüksiyon ve ameliyat sırasında bir burun konisi ile izofluran% 2,5) dir. Bu hayvan AAV tanecikleri dolaşan bir pozitif kontrol olarak görev yapacak. </li><li> Enjeksiyon ve 3 saat, 24 saat, 2 gün ve 3 gün ShH10 intravitreal enjeksiyonu sonrası veya intrapenile enjeksiyon (Şekil 1) önce sonra fare kuyruğu örnek kan. 10-20 ul heparin tüpler toplanır. Prosedürün tamamlanmasından sonra CO 2 inhalasyon fareler Kurban. </li><li> Seçtiğiniz ekstraksiyon kiti kullanılarak, kan örneklerinden genomik DNA ayıklayın. </li><li> Genomik DNA, PCR amplifikasyon yapın. Bu protokol için aşağıdaki primer çifti kullanın: GFP, 5&#39;-CGACACAATCTGCCCTTTCG-3 &#39;, 5&#39;-antisens CATGGACGAGCTGTACAAGGGA-3&#39; seziyorum. Aşağıdaki PCR programı gerçekleştirildi: 95 ° C&#39;de 2 dakika (1 döngü); 95 ° C, 45 sn, 55 ° C, 1 dakika, 72 ° C, 45 sn (30 döngü); 72 ° C&#39;de 5 dakika (1 döngü);4 ° C tutun. </li></ol> 5. İsteğe bağlı Evans Mavisi Yöntemi NOT: Evans mavisi yöntemi kullanılarak 10 retina içine kan damarlarından albümin kaçağı ölçerek damar geçirgenliği niceliğini. <ol><li> 5 um&#39;lik bir filtreden 5 ultrasonik banyoda dakika ve filtreleme için normal tuzlu su çözeltisi (6 mg / ml), sonikasyon içinde çözülerek Evans Mavisi boya hazırlanır. </li><li> (Adım 4.1, izofluran solumaktan) C57BL6J fareler anestezi refleksi, geri çekme refleksi ve uygun bir anesthetization gösteren kuyruk tutam yanıt doğrultma kaybını kontrol ve penil ven (Şekil 1) ile Evans mavisi (45 mg / kg) enjekte edilir. Farelerin pençeleri, namlu ve kulaklar, Evans mavisi enjeksiyonu takiben boya alımını ve dağılımını teyit açıkça mavi hale olmadığını kontrol edin. </li><li> Ketamin (50 mg / kg) ve ksilazin (10 mg / kg) ile, boyanın enjeksiyonundan sonra C57BL6J fareler 3 saat anestezisi ve rightin kaybını doğrulamakg doğru anesthetization gösteren refleks. Örnek intrakardiyak 500 heparin tüplerde kan ul bir sitrat tampon maddesi ile sol ventrikil yoluyla 2 dakika boyunca fareler serpmek (0.05 M, pH 3.5; sitrik asit 7.8 g damıtılmış su, 1 L sodyum sitrat ve 3.8 g çöz) vazokonstriksiyon önlemek için 37 ° C sıcaklığa daha önceden ısıtılmıştır. Fareler perfüzyon yoluyla kurban edilir. </li><li> Dikkatle perfüzyon, aydınlatmak ve sonra retina toplamak için bir işletim mikroskop altında her iki gözleri teşrih (Bölüm 3.2). Santrifüjlü bir buharlaştırıcıda Kuru retina gece boyunca, tartılmakta ve (100 xg) çalkalanarak 65 ° C&#39;de 18 saat formamid 100 ul ile retina inkübe edilerek Evans mavisi ekstrakte edin. 4 ° C&#39;de 2 saat süre ile 20,798.5 xg&#39;de 30K omega filtre tüpleri Santrifüj retina örnekler ve santrifüj kan örnekleri 4 ° C sıcaklıkta 15 dakika boyunca 20,798.5 xg&#39;de. </li><li> Absorbansı ölçmek için, her iki süpernatantlar kullanın. Absorbans en fo çıkarılarak her numunenin absorbansı belirlemek740 nm&#39;de absorbans minimum 620 nm mavi r Evans. Formamid Evans mavisi, standart bir eğriden plazma ve retinada Evans mavisi konsantrasyonu hesaplanır. Saatte kuru retina (plazma / g retina kuru ağırlık / saat ul) gram Evans mavisi mikrolitre içinde BRB geçirgenliği ifade eder. </li></ol> 6. İsteğe bağlı Floresein Anjiyografi Not: farelerdeki retinal kan damarlarından Kaçak sodyum fluoresein intraperitonal enjeksiyon ile görselleştirilebilir. <ol><li> C57BL6J fareler anestezi (izofluran solumaktan, adım 4.1) ve göz (neosynephrine ve mydriaticum) düşer Öğrencileri dilate. Anestezi altında iken kuruluğu önlemek için göze veteriner merhem kullanın. </li><li> Floresein anjiografi 0.1 ml steril tuzlu su içinde% 10 sodyum fluoresein karın 0,01-0,02 ml enjekte edilir. </li><li> Bir göz fundus kamera kullanarak enjeksiyon sonrası her iki gözün görüntüleri toplayın. Yanlısı sonra CO 2 solumaktan fareler Kurbanketencik. NOT: retinal damarlar anjiyografide görünür hale gelebilmesi için 20 sn gereklidir. Görüntüler floresein enjeksiyondan sonra 3-10 dk maksimum arasındaki yakalanan gerekir. kaçak noktalar (mevcut ise) 2.5 dakika sonra gözlenebilir. Daha sonra zaman noktalarında görüntü kalitesi nedeniyle sodyum floresein kısa bir süre analiz için kullanılan sonra elde edilen camsı, böylece sadece görüntü içine difüzyon kaybolur. </li></ol>

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The authors have nothing to disclose.

BRB kan ve retina arasındaki moleküller değişimini kontrol eder. Bu dökümü diyabetik retinopati veya yaşa ilişkin makula dejenerasyon (AMD) gibi çeşitli hastalıklarla ilişkilidir. Son zamanlarda geçirgen BRB görüntüleyen bir distrofin nakavt fare üzere retina adeno-ilişkili viral vektörler (AAV) tarafından aracılık edilen gen aktarımı için daha geçirgen hale göstermiştir. Ancak, BRB geçirgenlik AAV parçacıklar rağmen göz içine bu modelde göz bölmeye sınırlı kalmak enjekte. Sonuçl...

Müller hücreleri retinanın büyük glial bileşenidir. Morfolojik, onlar, vitreus ile temas radyal retina ve endfeet, yayılan ILM ve ikinci gizli bileşenleri karşı karşıya. ILM on farklı hücre dışı matris proteinleri (laminin Agrin, perlekan, nidojen, kollajen ve çeşitli heparin sülfat proteoglikanlar) oluşan bir taban zarıdır. Gelişimi sırasında, kendi varlığı ganglion hücrelerinin 1-3 retina histogenezisleri, optik akson navigasyon, ve hayatta kalmak için vazgeçilmezdir. Ancak, ILM yetişkin retinada önemsiz olduğunu ve cerrahi retina hasarı 4 neden olmadan belirli patolojilerin kaldırılabilir. Gen terapisi, bu membran intravitreal enjeksiyonu 5 ile AAVS kullanarak retinanın verimli iletimi için fiziksel bir bariyer haline gelir. Süreçlerini geniş arborization sayesinde, Müller hücreleri beslenme ve düzenleyici Suppo sağlamakoda sıcaklığı, retina nöronlarının ve vasküler hücrelerin hem de. Müller hücrelerinin de BRB 6 oluşumu ve bakım, retina homeostazının düzenlenmesinde rol oynamaktadır. Sıkı bağlantılar retina kapilar endotelial hücreler arasında, Müller hücrelerin, astrositlerin ve perisitler BRB oluştururlar. BRB diyabetik retinopati, retina ven tıkanıklığı ve solunum yolu hastalıkları gibi birçok hastalığın retina.In girmesini bazı maddeler önler, retinanın hipoksi BRB 7-9 ile sızıntı neden olur. Bu yırtılma vazojenik ödem, retina dekolmanı ve retina hasarına yol açan damar geçirgenliği bir artış ile ilişkilidir. Müller hücrelerinin sıkı hem BRB ve ILM bütünlük içinde önemli bir rol oynayan, kan damarları ve bazal membran ile ilişkilidir. Sonuç olarak, Müller glial hücreler etiketleme bu retina engellerin fiziksel durumunun çalışmaya özellikle önemlidir. Klasikortağı BRB geçirgenliği plazma albümini ile kovalent olmayan bir şekilde bağlanan Evans mavi boya, sistemik enjeksiyonundan oluşan Evans mavisi analizi kullanılarak ölçülür. Bu tahlil retina içine kan damarlarının 10 (Protokoller Bölüm 5&#39;e bakınız) albumin kaçağı (orta büyüklükte protein, ~ 66 kDa) ölçer. Alternatif olarak, vasküler sızıntı floresan sızıntı doğrulayan floresan retina anjiyografi ile görüntülendi olabilir (küçük molekülü, ~ 359 Da; Protokoller Bölüm 6) 11. Bununla birlikte, her iki yöntem küçük moleküller ve proteinlere BRB geçirgenlik değerlendirilmesini sağlar ancak ILM bütünlüğü hakkında bilgi vermemektedir. Bu nedenle, BRB geçirgenliğini incelemek için, biz, daha büyük moleküller (örneğin, AAV parçacıklar 25 nm çapında) için BRB geçirgenliği ile ilgili bilgi veren bir AAV esaslı yöntemi kullanılır. Nitekim, bizim yöntem öneririz, hangi kan AAV transgen varlığını tespit edebilir ~ 25 nm çapında parçacıkları olurkana sızmak mümkün. Bu yöntem, aynı zamanda patolojik durumlarda ILM ve hücre dışı matris proteinlerinin yapısı hakkında bilgi içerir. İki AAV varyantlar, çalışma için faydalıdır: AAV5 ve ShH10. Subretinally enjekte AAV5 fotoreseptör ve retina pigment epiteli 12 için doğal bir tropizm var ama sağlam ILM 5,13 ile yabani tip retina vitreus içine verildiğinde, dış retina genelinde alınamıyor. ShH10 özellikle nöron 14,15 fazla glial hücreler hedef için işlenmiş olan bir AAV varyantıdır. ShH10 seçici tehlikeye engelleri 16 retina artan verimlilik ile sağlıklı ve hastalıklı retina hem de Müller hücreleri etiketler. Immuhistochemistry ve kan-DNA analizi ile birleştiğinde bu viral araçlar retina engellerin devlet ve hastalık onların katılımı (Şekil 1) hakkında bilgi verir.

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using adeno-associated virus as a tool to study retinal barriers in disease

Müller hücrelerinin retina ana glial hücreler bulunmaktadır. Bunların son ayak, dış ve iç sınır membran (ILM) retinanın sınırlarını oluştururlar ve astrositler, perisitlere ve endotelyal hücreler ile bağlantılı olarak, kan-retina bariyerini (BRB) oluşturmaktadır. ILM retina ve vitreus boşluğuna arasında histolojik sınırı tanımlayan bir bazal membran gibi davranır ise BRB kan ve retina arasındaki malzeme taşıma sınırlar. Müller hücrelerinin etiketlenmesi bu hücreler BRB ve ILM ayrılmaz bir parçası olarak, retina bariyerinin fiziksel durumunu incelemek için özellikle önemlidir. BRB ve ILM Hem sık retina hastalığında değişmiş ve hastalık belirtileri sorumludur vardır. Böyle Evans mavisi deney veya floresein anjiyografi gibi brb bütünlüğünü incelemek için birkaç köklü yöntemler vardır. Ancak bu yöntemler BRB geçirgenlik t ölçüde bilgi vermemektedirnanometre aralığı içinde daha büyük moleküller, o. Ayrıca, böyle ILM gibi diğer retina engellerin durumuna ilişkin bilgi vermemektedir. Retina ILM ile birlikte BRB geçirgenliğini incelemek için, hastalık durumlarında ILM ve hücre dışı matris proteinlerinin durumunu gösteren ancak daha büyük moleküllere BRB geçirgenliği ile ilgili bilgi sağlayan bir AAV esaslı yöntemi kullanılır. İki AAV varyantlar, çalışma için faydalıdır: AAV5 ve ShH10. AAV5 Fotoreseptörlerin için doğal bir tropizm vardır ama ILM sağlam olduğunda (vahşi tip retina yani) vitreus içine verildiğinde, dış retina genelinde alınamıyor. ShH10 glial hücrelerine karşı güçlü bir tropizm vardır ve seçici sağlıklı ve hastalıklı retina hem de Müller glia etiketleyecektir. ShH10 ILM tehlikeye retina daha verimli gen teslim sağlar. Immunohistokimyası ve kan-DNA analizi ile birleştiğinde bu viral hastalığın araçlar retina engellerin durumuna da ışık tutacaktır.

Hayvan modeli ILM (- B Şekil 2A) yapısındaki düzensizlikler gösterir eğer biz ShH10 kullanarak Müller glial hücrelerin retina iletimini artış bekliyoruz. Örneğin, Dp71 yokluğunda göstermiştir, ShH10 hedefler özel olarak, fakat daha etkili Müller yabani tip farelere 16 (Şekil 2C - F) ile kıyaslandığında, bu fare çizgisinde ILM artan bir geçirgenlik gösteren intravitreal enjeksiyon ile glial hücreleri. RAAV...

Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Aracı Olarak Adeno-ilişkili virüs kullanarak Hastalığında Retina Engelleri Eğitim için

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

using-adeno-associated-virus-as-tool-to-study-retinal-barriers

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14.9K Görüntüleme.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Video: Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Rekombinant Adeno Associated Virus serotip 3 Vektörler Karaciğer Kanseri Hücreleri Yüksek Verimlilik İletimi

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Tıp

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Polikistik Böbrek Hastalığı Olan İnsanlar ve Fareler kromozom anomalileri Eğitim Spektral Karyotipik

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Pnömoni için Faydalı adjuvan Aracı Olarak Kemik Hastalıkları Manipülatif Tedavi

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

Bir Uygun Aracı Olarak Fundus Fotoğraf Epidemiyolojik Çalışmaların Kardiyovasküler Hastalıklar Risk Faktörleri için Mikrovasküler Cevapları Eğitim için

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

İnsan Videofloroskopik Kırlangıç ​​Eğitim Yöntemleri uyarlanması Algılama ve Mürin Hastalık Modelleri disfajiyi Characterize

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Bir rekombinant adeno-ilişkili viral vektörün stereotaktik enjeksiyon yoluyla Parkinson Hastalığı için bir alfa-sinüklein Bazlı Fare Modeli Geliştirme

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Demans çalışması için retina Imaging'i kullanma

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Virüs-aracılı teslim CRISPR kardiyak Gene farelerde düzenleme için Adeno ilişkili

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

The Dyspepsia Educational Tool As a Novel Aid in Dyspepsia Management

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from this development, as it enables patient engagement in self-management. Dyspepsia is a condition thought to arise from gastric and duodenal perturbations, brain-gut axis disturbances, and dietary factors. Behavioral interventions are a major part of dyspepsia treatment, hence patient engagement and motivation through education is essential. Protocols that describe the development process of such educational tools are scarce. We provide a methodology describing development of a dyspepsia educational tool. Assessment of users' needs is the first step, followed by a literature search. The content is developed based on the main themes and entered into a content management system, to build the program. Final adjustments are made after a pilot test of the tool. The presented protocol can be used as a guide for development of a digital dyspepsia educational tool or as a tool for similar situations.

This protocol describes the development process of a digital dyspepsia educational tool. Assessment of unmet needs and literature, content development, and building of the tool are presented. The methodology can be used as a guide for future development of digital educational tools.

Dispepsi yönetiminde yeni yardım olarak dispepsi eğitim aracı

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Küçük Hayvan Modellerinde Adeno İlişkili Virüs Vektörlerinin Subkonjonktival Uygulaması

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...