Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

Tüm protokoller geçerli nereye Kurumsal Hayvan Bakım ve Kullanım Komitesi politikalarına uygun yürütülmüştür. 1. Hazırlık <ol><li> % 75 ile sabitleme ortamı hazırlamak Dulbecco Modifiye Eagle Ortamı (DMEM),% 25 Hanks dengeli tuz çözeltisi (HBSS). kullanılana kadar -20 ° C&#39;de, iyi kısım ve mağaza karıştırın. </li><li> Fosfat tamponlu tuz (PBS) hazırlanması ve otoklavda sterilize edilir. Oda sıcaklığında çözüm saklayın. Deney (oyuk başına 5 mi) daha önce 37 ° C&#39;ye kadar PBS ısıtın. </li><li> % 10,% 20 ve PBS içinde% 30 sukroz hazırlayın. ayrı sterilizasyon için ayrı 0.22 mikron filtre üzerinden tüm çözümleri filtre. 4 ° C&#39;de çözüm kaydedin. </li><li> 90 mM&#39;lik bir konsantrasyona kadar PBS içinde histamin stok çözelti hazırlayın. 0.22 uM filtre üzerinde filtre edilerek çözelti sterilize edin. Kısım ve -80 ° C&#39;da, çözelti kaydedin. Birden fazla donma-ve-çözülme döngüleri kaçının. </li><li> deneyden önce PBS içinde taze% 4 paraformaldehid (PFA) yapın. Buz üzerine yerleştirin. Dikkat: Davlumbaz PFA tutun ve uygun koruyucu ekipman giyin. </li><li> stabilizasyon orta (retinanın başına 20 ml) çözülme. 100 U / ml bir son konsantrasyona kadar Penisilin-Streptomisin ekleyin. Deney öncesi bir inkübatörde 37 ° C&#39;ye kadar ortam (retinanın başına 15 mi) sıcak bölümü. buz üzerinde orta kalanı bırakın. </li><li> buz üzerinde HBSS (retinanın başına 10 ml) koyun. </li></ol> 2. Retina Organotipik Kültür <ol><li> Bir doku kültürü kaputu örnekleri aktarın. lensin etrafında keserek Göz yuvası açın. Bir fırça yardımıyla hafifçe retina üzerine çekerek olmadan vitreus mizah kaldırın. Yavaşça optik diski ortaya çıkarmak için bir fırça ile kesilmiş kenarından retina ayırın. optik bir ustura ile sinir ve retina serbest ağır. </li><li> Petri kabı içine retina aktarın ve dikkatle soğuk HBSS kullanarak bir kez retina durulayın. i üzerinde HBSS örnek yerleştirince. Adım 2.1 ve bu adımı tekrarlayarak gerektiği kadar retina parçalara ayır. </li><li> simetrik bir jilet ile ikiye retina kesti. Yavaşça sabitleme ortamının 3 ml 6 yuvalı bir plaka örnekleri transferi ile sabitleştirilir. % 5 CO2 içeren bir atmosfer ile bir inkübatör içinde 37 ° C&#39;de 30 dakika boyunca dengede izin verin. </li><li> Adım 2.3 de inkübasyon sırasında aşağıdaki reaktifler hazırlayın: sıcak ortamda istenen konsantrasyona (450 uM) histamin stok solüsyonu sulandırınız. 250 ng / ml &#39;lik bir nihai konsantrasyona kadar bir ortamda (argon altında saklanan) LXA4 çözülür. Not: LXA4 etkisini ölçerken, diğer yarısı LXA4 ile histamin kullanılır ise, tek bir retina yarısı tek başına histamin kullanılır. </li><li> dikkatli bir şekilde sabitleme ortamı aspire ve her bir (oyuk başına 3 mi) hazırlanan orta ekleyin. % 5 CO2 içeren bir atmosfer ile bir kuluçka makinesi içinde, 1 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> durulamabir zamanlar sıcak, steril PBS örnekler. </li></ol> 3. immün için Örnekleri hazırlayın <ol><li> plakalardan aspire PBS. % 4 PFA (oyuk başına 3 mi) ilave edilir ve oda sıcaklığında 15 dakika inkübe edilir. </li><li> Çabuk PFA aspire. bir zamanlar PBS kullanarak örnekleri durulayın. % 10 sakaroza örnekleri (oyuk başına 5 mi) aktarın ve 4 ° C&#39;de 2 saat ila 4 inkübe edilir. </li><li> % 30 sukroz (oyuk başına 5 mi) örnekleri aktarın ve 4 ° C&#39;de gece boyunca inkübe edilir. Çalışma donma orta yapmak için% 20 sukroz, iki parça ticari dondurma orta bir bölümünü karıştırın. kabarcıklar kaybolur hava kadar 4 ° C gecede veya daha uzun bırakın. </li><li> Çalışma dondurma ortamı örnekleri aktarın ve oda sıcaklığında 5 dakika için dengeleme sağlar. dikdörtgenler içine her retina (5 mm kabaca 3 mm) Trim. </li><li> Dikkatle silindirik bir kap (yaklaşık 1 cm çapında x 2 cm yükseklik) dev içerdiği çalışan dondurma ortama örnekleri aktarmakAlüminyum folyodan ised. retina dilimleri ve sıvı nitrojen içinde bunları dondurmak (Kesit üzerine retinanın bir kesitini sağlamaya yönelik) dik olduğundan emin olun. kullanılana kadar -80 ° C &#39;de bloklar kaydedin. </li><li> Bölüm her 14 mikron kalınlığında dilimler halinde bir kriyostat blok ve cam slaytlar bölümleri monte edin. kullanılana kadar -80 ° C &#39;de slaytlar saklayın. </li></ol> 4. immün <ol><li> bir kez% 5 sakaroz fosfat tamponu (SPB, PBS içinde% 5 sukroz, pH 7.4) ile bölümleri yıkayın. Oda sıcaklığında 1 saat boyunca (% 10 normal keçi serumu ve% 0.5 Triton X-100 ile% 5 SPB) blokaj tamponunda bölümleri inkübe edilerek spesifik olmayan bağlanma yerlerini bloke. </li><li> Uygun primer antikor ile (slaytlar üzerinde) bölümleri inkübe (1&#39;de seyreltilmiş GFAP ya MAP2&#39;nin: 500 bloklama tamponu içinde) 1 saat boyunca. çözüm aspire. % 5 SPB içinde üç kez yıkayın. endojen IgG boyama için, bu adımı atlayın. </li><li> İlgili secon ile bölümleri inkübe1 saat: dary antikorlar (bloke edici tampon içinde 800 Cyanine3 / FITC konjüge edilmiş eşek anti-fare / anti-tavşan IgG, 1 seyreltilmiş). </li><li> iyice% 5 SPB üç kez bölümleri yıkayın. orta montaj DAPI içeren ve lamelleri ile kaplama onları monte edilerek mikroskopi için örnekler hazırlayın. </li><li> Gruplar arasında, vb kalma süresi, lazer yoğunluğu, kazanç değeri olarak aynı parametreleri altında 20X merceğinden bir lazer konfokal mikroskop, yakalama görüntüleri kullanma. 488/525 nm olarak 561/785 nm olarak (Cyanine3 algılamak için) kırmızı kanal için, 408/447 nm olarak (DAPI tespit etmek) ve yeşil kanal için (FITC algılamak için) mavi kanal için uyarma / emisyon dalga boylarını ayarlamak . </li></ol> 5. Görüntü Analizi <ol><li> Aşağıdaki kriterlere 12&#39;ye göre sızdıran kan damarlarının yüzdesini ölçmek: sayısında bölümün düzlemi içinde endotel hücre çekirdekleri ile sadece kan damarları dahil; o ag gösterirse bir kan damarı sızan düşününkabı çevreleyen immün radient. </li><li> ifade seviyesini belirlemek için, ilgi alanları seçmek ve görüntü analizi yazılımı kullanılarak ortalama gri değeri ölçmek. </li><li> sürecin genişliğini ölçmek için, süreçler (örneğin dış çekirdek katmanı, onl) farklı olan bölümün bir alanı seçin. Sonra bu süreçler görünür ve sürekli olan bir optik alanı seçin. Mikroskobik ayarına göre ölçek çıtayı süreci boyunca bir çizgi çizin ve ölçümü yapmak. </li><li> sürecin devamlılığını analiz etmek için, ilgi seçkin bölgeleri, komşuluk varyans ile her pikseli değiştirerek Görüntüdeki kenarları artırır varyans denilen filtre uygulamak, otomatik olarak, kontrast ve parlaklık ayarı satırları saymak tarafından sayımı normale Konum. </li><li> İki kuyruklu Student t-testi kullanılarak istatistiksel anlamlara hesaplayın. * P &lt;0.05; ** P &lt;0.01; *** P &lt;0.001. Plot Mean olarak sonuç SEM ±. </li></ol>

<ol>
	<li>Cunha-Vaz, J., Bernardes, R., Lobo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-retinal+barrier.">Blood-retinal barrier.</a> J Ophthalmol. 21, 3 (2011).</li><li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-neural+barrier:+intercellular+communication+at+glio-vascular+interface.">Blood-neural barrier: intercellular communication at glio-vascular interface.</a> J Biochem Molec Biol. 39, 339-345 (2006).</li><li>Kumagai, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+transport+in+brain+and+retina:+implications+in+the+management+and+complications+of+diabetes.">Glucose transport in brain and retina: implications in the management and complications of diabetes.</a> Diabetes Metab Res Rev. 15, 261-273 (1999).</li><li>Runkle, E. A., Antonetti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-retinal+barrier:+structure+and+functional+significance.">The blood-retinal barrier: structure and functional significance.</a> Methods Molec Biol. 686, 133-148 (2011).</li><li>Takata, K., Hirano, H., Kasahara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+glucose+across+the+blood-tissue+barriers.">Transport of glucose across the blood-tissue barriers.</a> Int Rev Cytol. 172, 1-53 (1997).</li><li>Goncalves, A., Ambrosio, A. F., Fernandes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+claudins+in+blood-tissue+barriers+under+physiological+and+pathological+states.">Regulation of claudins in blood-tissue barriers under physiological and pathological states.</a> Tissue Barriers. 1, 24782 (2013).</li><li>Patton, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+image+analysis:+concepts,+applications+and+potential.">Retinal image analysis: concepts, applications and potential.</a> Prog Ret Eye Res. 25, 99-127 (2006).</li><li>Di Marco, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+dysfunction+in+the+pathogenesis+of+Alzheimer's+disease--A+review+of+endothelium-mediated+mechanisms+and+ensuing+vicious+circles.">Vascular dysfunction in the pathogenesis of Alzheimer's disease--A review of endothelium-mediated mechanisms and ensuing vicious circles.</a> Neurobiol Dis. 82, 593-606 (2015).</li><li>Nagele, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-reactive+autoantibodies+prevalent+in+human+sera+increase+intraneuronal+amyloid-beta(1-42)+deposition.">Brain-reactive autoantibodies prevalent in human sera increase intraneuronal amyloid-beta(1-42) deposition.</a> J Alzheimer's Dis: JAD. 25, 605-622 (2011).</li><li>Goldwaser, E., Acharya, N., Nagele, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+and+blood-brain+barrier+compromise:+A+mechanistic+link+between+vascular+disease+and+Alzheimer's+disease+subtypes+of+neurocognitive+disorders.">Cerebrovascular and blood-brain barrier compromise: A mechanistic link between vascular disease and Alzheimer's disease subtypes of neurocognitive disorders.</a> J Parkinsons Dis Alzheimer's Dis. 2, 10 (2015).</li><li>Horton, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+three-photon+microscopy+of+subcortical+structures+within+an+intact+mouse+brain.">In vivo three-photon microscopy of subcortical structures within an intact mouse brain.</a> Nat Photonics. 7, (2013).</li><li>Sedeyn, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+Induces+Alzheimer's+Disease-Like+Blood+Brain+Barrier+Breach+and+Local+Cellular+Responses+in+Mouse+Brain+Organotypic+Cultures.">Histamine Induces Alzheimer's Disease-Like Blood Brain Barrier Breach and Local Cellular Responses in Mouse Brain Organotypic Cultures.</a> Biomed Res Int. 2015, 937148 (2015).</li><li>Avdeef, A., Deli, M. A., Neuhaus, W., Di, L., Kerns, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Blood-Brain Barrier in Drug Discovery: Optimizing Brain Exposure of CNS Drugs and Minimizing Brain Side Effects for Peripheral Drugs. , 188 (2014).</li><li>Eigenmann, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+four+immortalized+human+brain+capillary+endothelial+cell+lines,+hCMEC/D3,+hBMEC,+TY10,+and+BB19,+and+optimization+of+culture+conditions,+for+an+in+vitro+blood-brain+barrier+model+for+drug+permeability+studies.">Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.</a> Fluids Barriers CNS. 10, 33 (2013).</li><li>Takeshita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+blood-brain+barrier+model+combining+shear+stress+and+endothelial+cell/astrocyte+co-culture.">An in vitro blood-brain barrier model combining shear stress and endothelial cell/astrocyte co-culture.</a> J Neurosci Methods. 232, 165-172 (2014).</li><li>Alberghina, M., Lupo, G., Anfuso, C. D., Moro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+transport+through+the+blood-retina+and+blood-brain+barrier+of+rat+visual+system+during+aging.">Palmitate transport through the blood-retina and blood-brain barrier of rat visual system during aging.</a> Neurosci Lett. 150, 17-20 (1993).</li><li>Hosoya, K., Yamamoto, A., Akanuma, S., Tachikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilicity+and+transporter+influence+on+blood-retinal+barrier+permeability:+a+comparison+with+blood-brain+barrier+permeability.">Lipophilicity and transporter influence on blood-retinal barrier permeability: a comparison with blood-brain barrier permeability.</a> Pharm Res. 27, 2715-2724 (2010).</li><li>Minamizono, A., Tomi, M., Hosoya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+dehydroascorbic+acid+transport+across+the+rat+blood-retinal+and+-brain+barriers+in+experimental+diabetes.">Inhibition of dehydroascorbic acid transport across the rat blood-retinal and -brain barriers in experimental diabetes.</a> Biol Pharm Bull. 29, 2148-2150 (2006).</li><li>Serlin, Y., Levy, J., Shalev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+pathology+and+blood-brain+barrier+disruption+in+cognitive+and+psychiatric+complications+of+type+2+diabetes+mellitus.">Vascular pathology and blood-brain barrier disruption in cognitive and psychiatric complications of type 2 diabetes mellitus.</a> Cardiovasc Psychiatry Neurol. 2011, 609202 (2011).</li><li>Kolb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+retina+works+-+Much+of+the+construction+of+an+image+takes+place+in+the+retina+itself+through+the+use+of+specialized+neural+circuits.">How the retina works - Much of the construction of an image takes place in the retina itself through the use of specialized neural circuits.</a> Am Sci. 91, 28-35 (2003).</li><li>Ikram, M. K., Cheung, C. Y., Wong, T. Y., Chen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pathology+as+biomarker+for+cognitive+impairment+and+Alzheimer's+disease.">Retinal pathology as biomarker for cognitive impairment and Alzheimer's disease.</a> J Neurol Neurosurgery Psychiatry. 83, 917-922 (2012).</li><li>Tan, Z., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta,+the+retina,+and+mouse+models+of+Alzheimer+disease.">Amyloid-beta, the retina, and mouse models of Alzheimer disease.</a> Am J Pathol. 176, 2055 (2010).</li><li>Serhan, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-resolving+lipid+mediators+are+leads+for+resolution+physiology.">Pro-resolving lipid mediators are leads for resolution physiology.</a> Nature. 510, 92-101 (2014).</li><li>Bucolo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+topical+indomethacin,+bromfenac+and+nepafenac+on+lipopolysaccharide-induced+ocular+inflammation.">Effects of topical indomethacin, bromfenac and nepafenac on lipopolysaccharide-induced ocular inflammation.</a> J Pharm Pharmacol. 66, 954-960 (2014).</li><li>Edelman, J. L., Lutz, D., Castro, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticosteroids+inhibit+VEGF-induced+vascular+leakage+in+a+rabbit+model+of+blood-retinal+and+blood-aqueous+barrier+breakdown.">Corticosteroids inhibit VEGF-induced vascular leakage in a rabbit model of blood-retinal and blood-aqueous barrier breakdown.</a> Exp Eye Res. 80, 249-258 (2005).</li></ol>

The authors have nothing to disclose.

In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

Artan miktarda kanıt vücut kan retina bariyerinin (BRB) 1-5 ve kan beyin bariyeri (KBB) 6,7 olan benzerliği varlığını destekler. BBB Uzlaşma sıkıca böyle deliryum 10 olarak nedensel ya da Alzheimer hastalığı (AH) gibi kronik nörodejeneratif hastalıklar tanısal belirteç olarak 8,9 ve akut koşulları ile bağlantılı olmuştur. potansiyel ilaç hedefleri için bu patolojilerin ve keşifler içine mekanistik anlayışlar genellikle beynin sınırlı erişilebilirlik ve ağ karışıklık tarafından engellenmektedir. Bu, in vivo görüntüleme 11, beyin Organotipik kültürü 12, birincil hücre kültürleri 13,14 ve ko-kültür sistemleri 15 gibi alternatifler oluşturulmuştur. Ancak bu modellerin çoğu hücreleri tanımlamak için özel araçlar, uzun deneysel dönemleri veya birden fazla belirteçler gerektirir. Fonksiyonel ve yapısal BBB ile BRB arasındaki benzerlikler yanı sıra dy arasında bir korelasyoniki sfunctions 16-19 iddia edilmiştir. Buna ek olarak, daha kolay erişim, iyi tanımlanmış hücre tipleri ve tabakalı yapı beyne bir pencere olarak iyi karakterize edilmiş retina izin vermiştir. BBB ve BRB yapısal ve işlevsel kimlikler detaylı karşılaştırılması gerekmektedir. Bununla birlikte, retina hastalıkları, özellikle de BRB ihlali da sıkı bir şekilde diyabet 18-19 ve AD 21,22 dahil olmak üzere çeşitli hastalıkların ilerlemesinin ile ilişkilendirilmiştir. Bu durumda, bir BRB disfonksiyon sistemi mekanizmasını tanımlamak için değil, aynı zamanda, potansiyel ilaç taranması için sadece kurmak ilgi çekicidir. Bu raporda, basit bir akut retinal kültürünü kullanarak bir protokol sağlayan BRB disfonksiyonu gelişti ve sunulmuştur. Artmış BBB geçirgenliği ve AD-benzeri patolojik değişiklikler histamin, bir pro-inflamatuar arabulucu 12 ile inkübe bir beyin Organotipik kültürü kurulmuştur. Bu nedenle, sunulan sistemde, histamin İçi olduBRB disfonksiyonu ikna etmek için ex vivo retina kültürüne ied. Böyle Mus musculus ve Bos Boğa gibi çeşitli türlerden gelen retinaları, test edilmiştir. Nedeniyle ticari kullanılabilirliği ve insan dokusuna benzediği için, taze domuz gözbebekleri burada bildirilen veri sağlamak için kullanılmıştır. Histamin ve / veya diğer ilaçlar ile inkübe edildikten sonra, retinalar immünoglobülin G (IgG), kan ana bileşenden biri olarak bir kaç protein 12 için immün değerlendirme için işleme alınmıştır; Glial fibriller asidik protein (GFAP), glial aktivasyon için iyi bilinen bir işaretleyici; ve mikrotübül-ilişkili protein 2 (MAP2), mikrotübül montaj için gerekli bir nörona özgü sitoskeletal proteini. Bundan başka, retinanın tabakalı yapı gibi genişliği ve süreklilik değişiklikler olarak Müller hücrelerinin ve gangliyon hücreleri proseslerinin ayrıntılı bir analizini sağlar. Böylece birkaç ek parametreler sonuçlarını değerlendirmek için kullanılabilirBRB ihlali erken bir aşamada ve aynı zamanda potansiyel tedavilerin ters etkilerini değerlendirmek için. kan damarlarının sızıntı (KZ), glial hücrelerin aktivasyonu ve nöronal hücrelerin hasar tepki: Bu protokol, ekranlı ilaçların potansiyel ters etkileri üç açıdan değerlendirilir. Çeşitli ölçümü yöntemleri, örneğin, ifade seviyesi immün bir süreç ve bir donanım filtresi tarafından gösterilen nöronal süreçlerin sürekliliği genişliği ölçüm yoğunluğu ile gösterilen, kullanılmaktadır. Daha iyi yöntemi göstermek için ve sonuçları yorumlamaya yardımcı olmak için, Lipoksin A4 (LXA4), endojen inflamatuar hasarına yanıt ve endotel disfonksiyonu 23 hafifletici sentezlenen bir bileşik, gösteri amaçlı seçilmiştir.

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an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

Biz histamin tarafından uyarılan BRB ihlal karşı koruyabilecek potansiyel tedaviler değerlendirmek için düşük maliyetli, zaman verimli ve kolay kullanımlı bir sistem sunuyoruz. IgG kontrol retina (Şekil 1A) damarlar içinde kısıtlı, ancak modelin başarıyla kuruldu doğrulayan histamin maruz (Şekil 1B) üzerine kan damarlarının, dışarı sızdırıyor. LXA4 sunulan sistemde...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Düşük maliyetli, kullanımı kolay ve güçlü bir sistem histamin tarafından uyarılan kan retina bariyer ihlali iyileştirecek potansiyel tedaviler değerlendirmek için kurulmuştur. Kan damarı sızıntı, Müller hücre aktivasyonu ve nöron sürekliliği bir potansiyel ilaç, Lipoksin A4 hasarı yanıtını ve ters değerlendirmek için kullanılır.

Tedavi Kan Retina Bariyer ihlali ve Potansiyel İlaçlar değerlendirilmesi için bir Akut Retina Modeli

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

an-acute-retinal-model-for-evaluating-blood-retinal-barrier-breach

Research

JoVE Journal

Medicine

7.1K Görüntüleme.  Rowan University.  Düşük maliyetli, kullanımı kolay ve güçlü bir sistem histamin tarafından uyarılan kan retina bariyer ihlali iyileştirecek potansiyel tedaviler değerlendirmek için kurulmuştur. Kan damarı sızıntı, Müller hücre aktivasyonu ve nöron sürekliliği bir potansiyel ilaç, Lipoksin A4 hasarı yanıtını ve ters değerlendirmek için kullanılır. 

Video: An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Akut Hepatit Model Uygulama: Görselleştirme ve Hepatik Mikrosirkülasyon Kan Akış ve Oksijen Tüketimi Analizi

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Tıp

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

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

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

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

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Bir oksijen-Glukoz Yoksunluk ve reoksijenizasyonun<em&gt; In Vitro</em&gt; İskemi-Reperfüzyon Modeli incelenmesi Kan-Beyin Bariyeri Disfonksiyon için

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

Retina Hastalıkları Değerlendirilmesi Nicel Otofloresans

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Tavşanlarda Subretinal Cerrahi Adım Protokolle Bir Adım

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Yöntem bir Vitro kan - beyin bariyerini modeli domuz beyin endotel hücreleri temel alan kurulması için geliştirilmiş

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

Gut-kan bariyerini ve karaciğer metabolizması Microbiota ürünlerin değerlendirilmesi için In Vivo yöntemi

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

Kemirgenlerde İskemik Retina Hastalıkları için Oksijen Kaynaklı Retinopati Modeli

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

İnsan Pluripotent Kök Hücrelerinden 3D Retinal Dokuların Türetilmesi için Retinal Organoid İndüksiyon Sistemi

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Dejeneratif Retina Hastalıkları için İnsan Dışı Primat Modelinde Retina Pigment Epitel Transplantasyonu

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...