Name: isolation of retinal arterioles for ex vivo cell physiology studies
Uploaded: 2018-07-14T15:00:00.000+00:00
Duration: 12 min 42 s
Description: Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

Bu raporda açıklanan protokoller geliştirilmesi hibe aşağıdaki finansman kuruluşları tarafından desteklenen: BBSRC (1/I026359/BB), görme (1429 ve 1822), juvenil diyabet araştırma Vakfı (2-2003-525), Wellcome Trust (074648/Z/04), için mücadele İngiliz kalp Vakfı (PG/11/94/29169), HSC R & D bölümü (STL/4748/13) ve MRC (MC_PC_15026).

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	<li>Kohner, E. M., Patel, V., Rassam, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+blood+flow+and+impaired+autoregulation+in+the+pathogenesis+of+diabetic+retinopathy.">Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy.</a> Diabetes. 44, 603-607 (1995).</li><li>Hafez, A. S., Bizzarro, R. L., Lesk, 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=Evaluation+of+optic+nerve+head+and+peripapillary+retinal+blood+flow+in+glaucoma+patients,+ocular+hypertensives,+and+normal+subjects.">Evaluation of optic nerve head and peripapillary retinal blood flow in glaucoma patients, ocular hypertensives, and normal subjects.</a> American Journal of Ophthalmology. 136, 1022-1031 (2003).</li><li>Curtis, T. M., Gardiner, T. A., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+lesions+of+diabetic+retinopathy:+clues+towards+understanding+pathogenesis?.">Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis?.</a> Eye (London). 23, 1496-1508 (2009).</li><li>Pournaras, C. J., Rungger-Br&#228;ndle, E., Riva, C. E., Hardarson, S. H., Stefansson, E. <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+retinal+blood+flow+in+health+and+disease).">Regulation of retinal blood flow in health and disease).</a> Progress in Retinal and Eye Research. 27, 284-330 (2008).</li><li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+retinal+vascular+patterns.+I.+Normal+architecture.">Studies of retinal vascular patterns. I. Normal architecture.</a> Archives of Ophthalmology. 64, 904-911 (1960).</li><li>Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+physiology+of+retinal+and+choroidal+arteriolar+smooth+muscle+cells.">Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.</a> Microcirculation. 14 (1), 11-24 (2007).</li><li>Puro, D. 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K., 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=TRPV2+Channels+Contribute+to+Stretch-Activated+Cation+Currents+and+Myogenic+Constriction+in+Retinal+Arterioles.">TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles.</a> Investigative Ophthalmology &amp; Visual Science. 57 (13), 5637-5647 (2016).</li><li>Guidoboni, 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=Intraocular+Pressure,+Blood+Pressure,+and+Retinal+Blood+Flow+Autoregulation:+A+Mathematical+Model+to+Clarify+Their+Relationship+and+Clinical+Relevance.">Intraocular Pressure, Blood Pressure, and Retinal Blood Flow Autoregulation: A Mathematical Model to Clarify Their Relationship and Clinical Relevance.</a> Investigative Ophthalmology &amp; Visual Science. 55 (7), 4105-4118 (2014).</li><li>Scholfield, C. N., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+cytosolic+calcium+regulation+among+different+microvascular+smooth+muscle+cells+of+the+rat+retina.">Heterogeneity in cytosolic calcium regulation among different microvascular smooth muscle cells of the rat retina.</a> Microvascular Research. 59 (2), 233-242 (2000).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nifedipine+blocks+Ca2++store+refilling+through+a+pathway+not+involving+L-type+Ca2++channels+in+rabbit+arteriolar+smooth+muscle.">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> Journal of Physiology. 532 (3), 609-623 (2001).</li><li>Tumelty, J., 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=Endothelin+1+stimulates+Ca2+-sparks+and+oscillations+in+retinal+arteriolar+myocytes+via+IP3R+and+RyR-dependent+Ca2++release.">Endothelin 1 stimulates Ca2+-sparks and oscillations in retinal arteriolar myocytes via IP3R and RyR-dependent Ca2+ release.</a> Investigative Ophthalmology &amp; Visual Science. 52 (6), 3874-3879 (2011).</li><li>Huynh, K. 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Dr. Joanna Kur an işçi-in Medtronic (Kardes sehirleri, Minnesota, ABD), şimdi onun mevcut çalışma değil çatışma burada sunulan ile.

Yukarıda açıklanan protokoller pratik gerektirir ama çok az sorun giderme adımları konusunda ulaşılabilir olmalıdır. Ortalama bir günde 6-8 kullanışlı arteriyoller izolasyonu elde etmek ve 3-4 başarılı deneyler ulaşmak. Karşılaşılan sorunlar, ancak, başarı oranlarını artırmaya yardımcı olmak için alınan bazı adımlar vardır. Zaman zaman biz bulduk, özellikle genç rats (< 8 hafta), kullanırken arteriyoller verimi düşük olabilir. Bu sorunu aşmak için her bir toz arasında retina dokusunun (10-30 s 500 x g) centrifuging içinde 1.12-1.14 adım öneririz. Bu kez damarları verimini artırmak için yardımcı olur ancak aynı zamanda hücre artıkları hazırlık içinde miktarını artırır.
Basınç myography çalışmalar kapları cannulating zaman arteriyoller yakın çatallanma site i ciddi herhangi bir yan dalları için dikkatli bir şekilde kontrol etmek önemlidir. Bu ortak bir neden kaçak ve basınç kaybı deneme sırasında temsil eder. [Ca2 +]ben iletişim kuralları ile ortaya çıkabilir ana konu yükleme boya düzeyidir. Zavallı boya yükleme düşük sinyal-gürültü oranı, normal Ca2 + homeostazı çalışmasının sonuçlarında aşırı yükleme sırasında yol açar. Ya bu sorunların olasılığını azaltmak için Retina homogenate küçük bir aliquot kaldırılabilir ve izole kaplar periyodik olarak yükleme iletişim kuralı sırasında kontrol. Girişim yama-kelepçe deneyler, gigaseal formasyonu başarı oranını olduğunda nasıl son derece bağımlı de bazal lamina sindirmek. Ne zaman başlangıçta bu protokol test veya yeni birçok enzim kullanarak bu enzim sindirim konsantrasyon/süresi ayarlamak gerekli olabilir. Dikkatle endotel ve düz kas katmanları ayrılması izleme yeterince bazal kaldırmak için yeterli sindirim sağlayacak laminal erişim kazanmak için. Dikkatli izlenmesi de gemi sıkmak başlar gibi meydana gelen aşırı sindirim, önlemek gereklidir. Bu durum ortaya çıkarsa, düz kas hücreleri sık sık düzeltme eki kelepçe kayıt için çok kırılgan. Enzimler uygularken DNaz eklemek önemlidir ben üzerinden kurtarılmış DNA iplikçiklerini kaldırılmasını sağlamak için yalıtım işlemi sırasında hücreler zarar görmüş. DNA parçalarının yapışkan ve Temizleme işlemi (adım 4.4), son aşamalarında arteriyol için uymaları forseps neden kez gemi kaybı sonuçlanan. Gemilerin temizlik Teknik olarak zor ve en yüksek büyütme oranını en iyi olası ucu çapının kapalı forseps (20 X) nazik süpürme hareketleri ile gerçekleştirilir. Temizleyicileri arasında laboratuvar rulo ile forseps temiz.
Vurgulanan daha önce bir anahtar motivasyon arkasında bu el yazması özetlenen protokolleri gelişimi daha iyi kan akımı retinal vasküler hastalıklar sırasında neden bozulur anlamak için oldu. En-in bizim iş bugüne Diyabetik göz hastalığı28,33üzerinde odaklanmıştır. Arteriyoller kemirgen modeller 1 bölümünde açıklanan yöntemleri kullanarak diyabetin Retina izole olabilir. Diyabetik hayvanlardan izole Retina arteriyoller üzerinde deney yakından tarafından damarları vivo içindedeneyimli hiperglisemik koşulları çoğaltmak denemek önemlidir. Bu nedenle, normalde yalıtım ve 25 mM deneysel çözümler D-glikoz seviyelerini yükseltmek. Vasküler Bodrum membranlar kalinlasma diyabet34,35sırasında Retina damarlarında iyi tanınan bir olgudur. Hayvanlar uzun süreli diyabet (> 1 ay hastalık süresi) kullanırken, artan enzim konsantrasyonları veya sindirim kez kez yama-kelepçe kayıt yöntemleri uygulamayı etkinleştirmek için gereklidir.
Ex vivo kullanmanın önemli bir sınırlama retinal vasküler Fizyoloji çalışması için Retina arteriyoller izole ve patofizyolojisi çevreleyen Retina neuropile kayıptır. Kaldırma Retina gliyal ve nöronal hücre hücre fizyolojisi çalışmaları retinal vasküler düz kas hücreleri için kolay erişim sağlar, ancak, gemilerin yanıt vazoaktif arabulucu olarak önemli ölçüde devamsızlık ve Retina varlığı değiştirebilirsiniz doku. Adenozin tri-fosfat (ATP), eylemler Örneğin, bu noktada iyi bir örnek sağlar. İçinde izole kaplar36sağlam bir daralma Retina arteriyoller, ATP Tetikleyicileri eklenmesi sıçan, sağlam bir neuropile varlığı ise37damarlar dilate. Bu nedenle, mümkün olan her yerde, biz genellikle izole arteriyol hazırlıklarımız Retina bütün bağlar- ex vivo ve in vivo ölçümleri damar çapı ve kan akışı16,37 kullanarak anahtar bulgular doğrulamayı deneyin . Belirtmek gerekirse, küçük arteriyoller ve kılcal domuz Retina bütün periosteum ex vivo38,39' eğitimi için yeni yöntemler son zamanlarda ortaya çıkmıştır. Böyle hazırlıkları büyük ölçüde Retina neuropile Retina arterioler ve kapiller sesi nasıl düzenler ve nasıl nöronal aktivite retina Retina haemodynamics değişimler modüle anlayışımızı geliştirmek muhtemeldir.
Bu makalede açıklanan yordamlar anlama arterioler düz kas hücre fizyolojisi izole sıçan retina arteriyoller kullanımına odaklanmış olsa da, şu anda protokolleri de endotel hücre işlevinde çalışmanın etkinleştirmek için gelişmekte olan Bu gemiler. Ön çalışma, biz Ca2 + görüntüleme ve çalışmalar kayıt patchclamp mükellef bulunmaktadır uygun endotel hücre tüpleri vermeye Retina arterioler segmentlerinin enzimatik sindirim değiştirilmesinde başarılı olmuştur. Cannulation izole arteriyoller intraluminal teslim uyuşturucu sağlayarak her iki ucunda, gelecekte de bu gemiler araştırılması etkinleştir endotel bağımlı vasodilatory yanıt-e doğru olabilir.

Retinada kan akışını nasıl denetlenir en önemli noktadır önemli bir hedefi, anormal kan akımı görme tehdit eden çeşitli patogenezinde karıştığı olmuştur beri retina hastalıkları1,2,3, 4. İç retina sinir hücreleri ve Gliyal hücreler malzemeleri, retina dolaşım kan retina damarları ve kılcal damarlar geçen Retina venüller ve sonunda damarlar5arteriyoller tümünün bulunduğu bir bitiş arter düzenleme vardır. Kan akımı retina Retina arter ve arteriyoller sesi hem de retinal kapiller ve sonrası kapiller venüller6,7, duvarlarında yer alan perisitlerden contractile etkinliği tarafından düzenlenmiştir 8. retinal vasküler sesi kontrolünü karmaşıktır ve girişlerden dolaşım sistemi ve çevresindeki Retina dokusu, moleküller ve hormonlar, dolaşan kan gazı da dahil olmak üzere bir dizi tarafından modüle ve vazoaktif maddeleri yayınladı retinal vasküler endotel ve macroglia9,10,11. Retina arteriyoller retina küçük atardamar dallarında ve vasküler düz kas hücre tek bir tabaka oluşur ve bir iç astar boyuna endotel hücreleri12,13, düzenlenen 14. Bu gemiler vasküler direnç retina dolaşım içinde ana site oluşturmak ve bu nedenle retinal kan akımı yerel kontrol önemli bir rol oynamaktadır. Retina arteriyoller genişletici veya vasküler düz kas contractility10,15,16değişikliklerden aracılı onların luminal çapı constricting tarafından retina kılcal kan akışını düzenler. Moleküler mekanizmalar hangi Retina arteriyoller düzenleyen Retina perfüzyon bu nedenle nerede arterioler düz kas hücreleri erişilebilir ve koşulları olabildiğince fizyolojik olarak yakın Okulu'nu hazırlıklar gerektirir anlama.
Ex vivo hazırlıklar izole Retina kan damarlarının ederken hala onların işlevsellik ve altta yatan endotel ile Birleştiricisi koruyarak vasküler düz kas hücreleri erişim sağlar. İzole gemileri kullanan bugüne kadar en çalışmaları büyük sığır veya domuz arteryel gemilerin üzerinde (60-150 µm) odaklanmıştır. Bunlar piyasada bulunan tel veya vasküler düz kas hücre contractile mekanizmaları17,18farmakolojik sorgulama etkinleştirmek için basınç myograph sistemleri monte edilebilir. Böyle hazırlıkları büyük ölçüde normal şartlar altında retinal vasküler fizyoloji ile ilgili bilgilerimizi katkıda bulunmuştur. Kaç çalışmalar Retina arteriyoller küçük laboratuvar hayvan--dan onların daha küçük çapta izole kullandık (~ 8-45 µm) geleneksel myography sistemleri19,20,21,22bunların kullanımı engeller. Önemli bir avantaj, ancak, küçük Laboratuvar hayvanlarının gemileri genetik olarak değiştirilmiş, transgenik ve retina hastalığı modelleri geniş durumu kullanmaktır. Küçük Laboratuvar hayvanlarının da vivo içinde müdahale çalışmaları için daha fazla mükellef bulunmaktadır.
Burada izole ve sıçan retina arteriyoller basınç myography deneyleri için cannulating için basit iletişim kuralları açıklar. Bu gemiler kullanarak Ca2 + görüntüleme ve Elektrofizyoloji iletişim kuralları da ayrıntılı olarak açıklanmıştır. Bunlar daha fazla vasküler düz kas contractility ve kan akımı retina Yönetmeliği kazandırabileceğini.

<table><tbody><tr><td>Beakers</td><td>Fisherbrand</td><td>15409083</td><td>Or any equilavent product</td></tr><tr><td>Curved Scissors</td><td>Fisher Scientific</td><td>50-109-3542</td><td>Or any equilavent product</td></tr><tr><td>Disposable plastic pipette/ transfer</td><td>Sarstedt</td><td>86.1174</td><td>6mL is best but other sizes are acceptable; remove tip to widen aperture</td></tr><tr><td>Dissecting microscope</td><td>Brunel Microscopes LTD</td><td></td><td>Or any equilavent product</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #5 14095</td><td>Any equivalent fine forceps</td></tr><tr><td>Pasteur pipette</td><td>Fisherbrand</td><td>11546963</td><td>Fire polished to reduce friction but not enough to narrow the tip</td></tr><tr><td>Petri dish</td><td>Sigma-Aldrich</td><td>P7741</td><td>Or any equilavent 10cm product</td></tr><tr><td>Pipette teat</td><td>Fisherbrand</td><td>12426180</td><td>Or any equilavent product</td></tr><tr><td>Purified water supply</td><td>Merek</td><td>Milli-Q Integral Water Purification System</td><td>Or any equilavent system</td></tr><tr><td>Round bottomed test tube</td><td>Fisherbrand</td><td>14-958-10 B</td><td>5 mL; any equilavent product</td></tr><tr><td>Seratted forceps</td><td>Fisher Scientific</td><td>17-467-230</td><td>Or any equilavent product</td></tr><tr><td>Single edge blades</td><td>Agar Scientific</td><td>T585</td><td>Or any equilavent product</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>184</td><td>Or any pliable surface&amp;nbsp;</td></tr><tr><td>Testtube rack</td><td>Fisherbrand Derlin</td><td>10257963</td><td>Or any equilavent product</td></tr><tr><td>Pressure myography</td><td></td><td></td><td></td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>x2 (or any equilavent product)</td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software&amp;nbsp;</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Analysis plugin</td><td>Myotraker&amp;nbsp;</td><td>https://doi.org/10.1371/journal.pone.0091791.s002</td><td>Custom plugin for imageJ Freely available for download</td></tr><tr><td>Cannulation pipette glass</td><td>World Percision instruments</td><td>TW150F-4</td><td>Or any equilavent product</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fluo-4-AM</td><td>Thermo Fisher Scientific</td><td>F14201</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #7 14097</td><td>Any equivalent fine forceps</td></tr><tr><td>Fura-2-AM</td><td>Thermo Fisher Scientific</td><td>F1221</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Helper pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Manometer</td><td>Riester</td><td>LF1459</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W558818</td><td>75&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Or any equilavent product</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Video capture software</td><td>Hypercam</td><td>version 2.28.01</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Patch clamping</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2411</td><td>3 mg disolved daily in 50 &amp;mu;L of DMSO (sonicate to dissolve)</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200a</td><td>Or any equilavent product</td></tr><tr><td>Analogue digital converter</td><td>Axon</td><td>Digidata 1440A</td><td>Or any equilavent product</td></tr><tr><td>Collagenase Type 1A</td><td>Sigma-Aldrich</td><td>C9891&amp;nbsp;</td><td>0.1mg/mL in LCH</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>DNAse I</td><td>Millipore</td><td>260913</td><td>Working stock 1 MU mL-1 (10 MU diluted in 10 mL LCH)&amp;nbsp;</td></tr><tr><td>Faraday cage</td><td>Custom made</td><td></td><td>Any equilavent commerical product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Patching software</td><td>Molecular Devices</td><td>Pclamp v10.2</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Protease Type XIV</td><td>Sigma-Aldrich</td><td>P5147&amp;nbsp;</td><td>0.01mg/mL in LCH</td></tr><tr><td>Single channel pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>Whole cell pipette glass</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>x40 lens</td><td>Nikon</td><td>40x/0.55, WD 2.1, &amp;infin;/1.2</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt; imaging</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Acquisition software&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Acquisition Engine V1.1.5</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software for confocal imaging</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Confocal microscope</td><td>Leica Geosystems</td><td>SP5&amp;nbsp;</td><td>Or any equilavent product; confocal</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TE2000</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Monochromator&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Optoscan</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Software for confocal microscope</td><td>Leica Geosystems</td><td>LAS-AF version 3.3.</td><td>Or any equilavent product; confocal</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x100 lens</td><td>Nikon</td><td>x100 N.A. 1.3 oil</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Leica Geosystems</td><td>HCX PL FLUOTAR, 20x/0.50, &amp;infin;/0.17/D</td><td>Or any equilavent product; confocal</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x63 lens</td><td>Leica Geosystems</td><td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &amp;infin;/0.17/E</td><td>Or any equilavent product; confocal</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>&lt;strong&gt;Pipettes&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Specifications and settings for fabrication of pipettes for experimentation&lt;/strong&gt;</td></tr><tr><td>Helper pipette</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;10 M&amp;Omega;</td></tr><tr><td>Cannulation pipette</td><td>World Percision instruments</td><td>TW150F-4</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 290&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 58&lt;br/&gt; Time: 150&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 3-10 &amp;mu;m&lt;br/&gt; Polishing: no&lt;br/&gt; Final Resistance: &amp;lt;1 M&amp;Omega;</td></tr><tr><td>On-cell patching</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;5 M&amp;Omega;</td></tr><tr><td>Whole-cell patching</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 296&lt;br/&gt; Pressure: 200&lt;br/&gt; Heat: 287&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 50&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 2-3 &amp;mu;m&lt;br/&gt; Polishing: helpful but not necessary&lt;br/&gt; Final Resistance: 1-2 M&amp;Omega;</td></tr></tbody></table>

isolation of retinal arterioles for ex vivo cell physiology studies

Retina bir önemli kan akımı gerektirir yüksek metabolik olarak aktif bir dokudur. Photoreceptors choroidal damarları tedarik ederken iç retina retina dolaşım destekler. Retina perfüzyon değişiklikler diabetik retinopati dahil olmak üzere çok sayıda görüş tehdit bozuklukları için katkıda bulunmak, glokom ve Retina şube occlusions damar. Moleküler mekanizmalar yoluyla retina ve nasıl bunlar göz hastalığı sırasında değişmiş kan akışını kontrol dahil anlamak bu koşullar tedavisi için yeni hedefler tanımlaması neden olabilir. Retina arteriyoller ana direnç damarlarının Retina ve sonuç olarak, değişiklikleri luminal çapı ile retina hemodinami düzenlenmesinde anahtar bir rol oynamak. Son yıllarda, çeşitli hücre fizyolojisi çalışmaları da dahil olmak üzere uygulamalar için uygun olan arteriyoller sıçan retina dan izole için yöntemler geliştirdik. Bu hazırlık nasıl kan akımı retinada kontrol edilir ve bize bazı göz hastalığı sırasında oluşan anahtar değişiklikleri tanımlamaya izin verdi yeni anlayışlar vermeye başlamış durumda. Bu makalede, biz sıçan retina arteriyoller yalıtım için yöntemleri tarif ve yama-kelepçe Elektrofizyoloji, kalsiyum görüntüleme ve baskı myography çalışmalar bunların kullanımı için iletişim kuralları içerir. Bu gemiler de mükellef kullanılmak üzere PCR-, western blot ve immünhistokimya tabanlı çalışmalar bulunmaktadır.

Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Bu el yazması arteriyoller elektrofizyolojik, kalsiyum görüntüleme ve baskı myography çalışmalarda kullanılan fare retina üzerinden yalıtım için basit bir protokol açıklar.

Retina arteriyoller yalıtım Ex Vivo Hücre fizyolojisi çalışmaları için

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

isolation-of-retinal-arterioles-for-ex-vivo-cell-physiology-studies

Research

JoVE Journal

Biology

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

10.2K Görüntüleme.  Queen's University of Belfast. Bu el yazması arteriyoller elektrofizyolojik, kalsiyum görüntüleme ve baskı myography çalışmalarda kullanılan fare retina üzerinden yalıtım için basit bir protokol açıklar.

Video: Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Akış Sitometresi ile Primer Sıçan Retinalı Ganglion Hücrelerinin (RGC) İzolasyonu

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Biyomühendislik

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Yeni Nesil Dizileme için Murin Retinal Endotel Hücrelerinin İzolasyonu

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Gelişim Biyolojisi

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Fare Beyin Gemiler saflaştırılması

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Nörobilim

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

İzolasyon ve Serebral Parankimal arteriollerin kanülasyonu

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Fare retina arteriyoller Ex Vivo reaktivite ölçümü için hazırlık adımları

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Tıp

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

Sağlam Retina Pigment Epitel ile Organotipik Fare Retina Eksplantlarının Uzun Süreli, Serumsuz Ekimi

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Proliferatif Olmayan ve Proliferatif Retinopatilerle Farelerin Tüm Mount Retinalarında Vasküler Parametrelerin Nicelemesi

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Retinal Nörovasküler Hastalıkları İncelemek için Ex Vivo Modeli Olarak Yetişkin Fare Retinasının Retinal Eksplantı

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retina Cryo-kesitler, Bütün bağlar ve mikrovasküler perisitlerden immunohistokimyasal görselleştirme hipotonik izole damarlara hazırlıkları

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Biyoloji

Diyabetik Retinopati Patogenez Çalışması için Yeni Retina Koruma Testi

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

Yazar Spotlight: Retina Damar Esnekliğini ve Hastalığın İlerlemesini Anlamak

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

Farelerde Retina Damar Sisteminin Diyabete Bağlı Ölümden Korunmasını Saptamak İçin Bir Test

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...