Name: in vivo vascular permeability detection in mouse submandibular gland
Uploaded: 2022-08-04T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

Bu çalışma, Çin Ulusal Doğa Bilimleri Vakfı (hibeler 31972908, 81991500, 81991502, 81771093 ve 81974151) ve Pekin Doğa Bilimleri Vakfı (hibe 7202082) tarafından desteklenmiştir.

Tüm deneysel prosedürler Pekin Üniversitesi Sağlık Bilimleri Merkezi, Hayvan Araştırmaları Etik Kurulu tarafından onaylanmış ve Laboratuvar Hayvanlarının Bakımı ve Kullanımı Kılavuzu'na (NIH Yayın No. 85-23, gözden geçirilmiş 1996) uyulmuştur. Bu çalışmada 8-10 haftalık yaş grubundaki erkek vahşi tip (WT) fareler kullanılmıştır. Deney hayvanları, ağrılarını ve rahatsızlıklarını en aza indirmek için dikkatlice tedavi edildi.
1. Hayvan prosedürle...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proper+role+of+nerves+in+salivary+secretion:+A+review.">The proper role of nerves in salivary secretion: A review.</a> Journal of Dental Research. 66 (2), 387-397 (1987).</li><li>Berndt, P., 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=Tight+junction+proteins+at+the+blood-brain+barrier:+Far+more+than+claudin-5.">Tight junction proteins at the blood-brain barrier: Far more than claudin-5.</a> Cellular and Molecular Life Sciences. 76 (10), 1987-2002 (2019).</li><li>Cong, X., 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=Disruption+of+endothelial+barrier+function+is+linked+with+hyposecretion+and+lymphocytic+infiltration+in+salivary+glands+of+Sj&#246;gren's+syndrome.">Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sj&#246;gren's syndrome.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1864 (10), 3154-3163 (2018).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+Multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: Multiphoton microscopy in the biosciences.</a> Nature Biotechnology. 21 (11), 1369-1377 (2003).</li><li>Masedunskas, A., Sramkova, M., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostasis+of+the+apical+plasma+membrane+during+regulated+exocytosis+in+the+salivary+glands+of+live+rodents.">Homeostasis of the apical plasma membrane during regulated exocytosis in the salivary glands of live rodents.</a> Bioarchitecture. 1 (5), 225-229 (2011).</li><li>Masedunskas, A., 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=Role+for+the+actomyosin+complex+in+regulated+exocytosis+revealed+by+intravital+microscopy.">Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (33), 13552-13557 (2011).</li><li>Balda, M. S., 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=Functional+dissociation+of+paracellular+permeability+and+transepithelial+electrical+resistance+and+disruption+of+the+apical-basolateral+intramembrane+diffusion+barrier+by+expression+of+a+mutant+tight+junction+membrane+protein.">Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.</a> The Journal of Cell Biology. 134 (4), 1031-1049 (1996).</li><li>Enis, D. R., 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=Induction,+differentiation,+and+remodeling+of+blood+vessels+after+transplantation+of+Bcl-2-transduced+endothelial+cells.">Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (2), 425-430 (2005).</li><li>Wang, X., 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=Application+of+digital+subtraction+angiography+in+canine+hindlimb+arteriography.">Application of digital subtraction angiography in canine hindlimb arteriography.</a> Vascular. 30 (3), 474-480 (2022).</li><li>Trani, M., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+in+the+control+of+vascular+permeability:+vascular+endothelial-cadherin+and+other+players.">New insights in the control of vascular permeability: vascular endothelial-cadherin and other players.</a> Current Opinion in Hematology. 22 (3), 267-272 (2015).</li><li>Viazzi, F., 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+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertension Research. 31 (5), 873-879 (2008).</li><li>Scheppke, L., 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+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> The Journal of Clinical Investigation. 118 (6), 2337-2346 (2008).</li><li>Blanchet, M. R., 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=Loss+of+CD34+leads+to+exacerbated+autoimmune+arthritis+through+increased+vascular+permeability.">Loss of CD34 leads to exacerbated autoimmune arthritis through increased vascular permeability.</a> Journal of Immunology. 184 (3), 1292-1299 (2010).</li><li>Egawa, G., Ono, S., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging+of+vascular+permeability+by+two-photon+microscopy.">Intravital Imaging of vascular permeability by two-photon microscopy.</a> Methods in Molecular Biology. 2223, 151-157 (2021).</li><li>Vestweber, D., Wessel, F., Nottebaum, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+the+regulation+of+leukocyte+extravasation+and+vascular+permeability.">Similarities and differences in the regulation of leukocyte extravasation and vascular permeability.</a> Seminars in Immunopathology. 36 (2), 177-192 (2014).</li><li>Schulte, D., 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=Stabilizing+the+VE-cadherin-catenin+complex+blocks+leukocyte+extravasation+and+vascular+permeability.">Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability.</a> The EMBO Journal. 30 (20), 4157-4170 (2011).</li><li>Uhl, B., 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=A+novel+experimental+approach+for+in+vivo+analyses+of+the+salivary+gland+microvasculature.">A novel experimental approach for in vivo analyses of the salivary gland microvasculature.</a> Frontiers in Immunology. 11, 604470 (2020).</li></ol>

Endotel bariyer fonksiyonunun idamesi ve düzenlenmesi vasküler homeostaz için gereklidir. Endotel hücreleri ve bunların hücreler arası kavşakları, vasküler bütünlüğün korunmasında ve kontrol edilmesinde kritik bir rol oynamaktadır12. Kan akımının kayma kuvveti, büyüme faktörleri ve inflamatuar faktörler vasküler geçirgenlikte değişikliklere neden olabilir ve böylece hipertansiyon, diyabet ve otoimmün hastalıklar gibi sistemik hastalıkların ortaya çıkmasına ve ge...

Çeşitli tükürük bezleri, öncelikle enfeksiyonlara karşı ilk savunma hattı görevi gören ve sindirime yardımcı olan tükürük üretir, böylece ağız ve genel sağlıkta önemli bir rol oynar1. Kan akışı, tükürük bezi sekresyonu için çok önemlidir, çünkü sürekli olarak birincil tükürüğü oluşturan su, elektrolitler ve moleküller sağlar. Sıkı bağlantı (TJ) kompleksi tarafından düzenlenen endotel bariyer fonksiyonu, suya, çözünürlere, proteinlere ve hatta dolaşımdaki kan damarlarından tükürük bezi dokularına doğru hareket eden hücrelere oldukça geçirgen olan kılcal damarların geçirgenliğini kesin ve hassas bir şekilde sınırlar 2,3. Daha önce kolinerjik bir uyarana yanıt olarak endotel TJ'lerinin açılmasının tükürük sekresyonunu kolaylaştırdığını, oysa endotel bariyer fonksiyonunun bozulmasının Sjögren sendromunda submandibuler bezlerde (SMG'ler) hiposekresyon ve lenfositik infiltrasyon ile bağlantılı olduğunu bulmuştuk4. Bu veriler, çeşitli tükürük bezi hastalıkları ile ilgili endotel bariyer fonksiyonunun katkısına yeterince dikkat edilmesi gerektiğini göstermektedir.
İki fotonlu lazer taramalı mikroskop, bozulmamış dokudaki hücrelerin dinamiklerini in vivo olarak gözlemlemek için güçlü bir araçtır. Bu tekniğin avantajlarından biri, yakın kızılötesi ışığın (NIR), numuneler NIR tarafından uyarıldığında görünür veya ultraviyole ışıktan daha derin doku penetrasyonuna sahip olması ve uygun koşullar altında dokularda belirgin ışık hasarına neden olmamasıdır 5,6. Gerçekten de, tükürük bezleri, yüzey asinar hücrelerinin bez yüzeyinden 7,8 ila 30 μm uzaklıkta olduğu çok homojen ve yüzeysel bir dokudur. İntravital konfokal mikroskopinin, canlı fare tükürük bezlerinde ekzokrin sekresyonu ve aktin sitoiskeletini hücrealtı çözünürlükte 8'de inceleyebileceği gösterilmiştir. Bununla birlikte, iki fotonlu lazer taramalı mikroskopi, sadece geleneksel konfokal mikroskopinin avantajına sahip olmakla kalmaz, aynı zamanda daha derin doku ve görüntüyü daha net bir şekilde tespit etmek için de kullanılabilir. Burada, sıklıkla parasellüler geçirgenlik izleyicileri olarak kullanılan ve farklı boyutlarda avantaja sahip olan floresan etiketli dekstrans, TJ gözenek9'un büyüklüğünü test etmek için kullanılabilir. Bu çalışmada, fare SMG'lerinde endotel bariyer fonksiyonunun yerinde değerlendirilmesi için intravital gerçek zamanlı iki foton lazer taramalı mikroskopi tekniği oluşturulmuştur. Fare SMG'lerinde in vivo vasküler geçirgenlik tespiti için her çalışma adımı mevcut protokolde açıklanmıştır. İşte fare SMG kanal ligasyon modelinde endotel bariyer fonksiyonunu tespit etme örneği.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-photon microscope (TCS-SP8 DIVE)</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 kDa FITC-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>70 kDa rhodamine B-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>R9379</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt tissue separation nickel</td>
			<td>Bejinghuabo Company</td>
			<td>NZW28</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable sterile syringe</td>
			<td>Zhiyu Company</td>
			<td></td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>0253316</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Leica Application Suite X software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline 1x</td>
			<td>Servicebio</td>
			<td>G4207-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Bejinghuabo Company</td>
			<td>M286-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>JITIAN Bio</td>
			<td>JT0781</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo vascular permeability detection in mouse submandibular gland

Tükürük ağız ve genel sağlıkta önemli bir rol oynar. Kan damarlarının sağlam endotel bariyer fonksiyonu tükürük sekresyonunu sağlarken, endotel bariyer disfonksiyonu birçok tükürük bezi salgı bozukluğu ile ilişkilidir. Bu protokol, fare submandibuler bezlerinde (SMG) endotel sıkı kavşakların (TJ'ler) işlevini değerlendirmek için bir in vivo parasellüler geçirgenlik tespit yöntemini tanımlamaktadır. İlk olarak, farelerin açısal damarlarına farklı moleküler ağırlıklara (4 kDa, 40 kDa veya 70 kDa) sahip floresan etiketli dekstrans enjekte edildi. Daha sonra, tek taraflı SMG diseke edildi ve iki fotonlu lazer tarama mikroskobu altında özelleştirilmiş tutucuya sabitlendi ve daha sonra kan damarları, acini ve kanallar için görüntüler yakalandı. Bu yöntem kullanılarak, farklı büyüklükteki izleyicilerin kan damarlarından acini'nin bazal taraflarına ve hatta asinar epitelinin üzerinden kanallara gerçek zamanlı dinamik sızıntısı, fizyolojik veya patofizyolojik koşullar altında endotel bariyer fonksiyonunun değişimini değerlendirmek için izlendi.

Protokolü takiben, tek taraflı SMG ısmarlama bir tutucuya bağlandı ve bez, nefes almanın hareket artefaktlarına neden olmasını önlemek için fare gövdesinden mümkün olduğunca uzak tutuldu. Kan damarlarındaki kırmızı kan hücrelerinin (siyah noktalar) hızlı akışı mikroskop altında gözlendi. Bir oküler lens altında doku alanını bulduktan sonra, mikroskop yazılımını manipüle etmek için geçiş yapılmalıdır. Kontrol grubunda, her iki izleyici de fare SMG'sinin kan damarlarında mevcuttu....

Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Bu protokolde, submandibuler bezin (SMG) endotel bariyer fonksiyonu, iki fotonlu lazer taramalı mikroskop altında test hayvanı modellerinin açısal damarlarına in vivo olarak farklı moleküler ağırlıklı floresan izleyiciler enjekte edilerek değerlendirilmiştir.

İn Vivo Fare Submandibuler Bezinde Vasküler Geçirgenlik Tespiti

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

Bu protokol, fare submandibuler bezlerindeki sıkı endotel kavşaklarının işlevini değerlendirmek için in vivo parasellüler geçirgenlik tespit önlemini açıklamaktadır. Bununla birlikte, iki foton lazer tarama mikroskobu, sadece geleneksel konfokal mikroskopinin avantajına sahip olmakla kalmaz, aynı zamanda daha derin doku ve görüntüyü daha net bir şekilde tespit etmek için de kullanılabilir. Bu deney, farklı dokuların vasküler geçirgenliğini, özellikle de hayvanların yüzey dokularını ve organlarını değerlendirmek için iyi bir yöntem ölçüsü sağlar.Geçirgenlik testi için floresan sinyalleri arasındaki paraziti en aza indirmek için floresein, izotiyosiyanat, etiketli dekstran ve farklı uyarma emisyon spektrumlarına sahip dekstran etiketli Rhodamine B gibi uygun izleyicileri seçerek başlayın. İzleri steril fosfat tamponlu salin halinde mililitre stoklar başına 100 miligram halinde seyreltin ve ışıktan korunan alikotları eksi 20 santigrat derecede saklayın. 8 ila 10 haftalık erkek vahşi tip fareyi anestezi altına aldıktan sonra, göz küresinin bir tarafının hafifçe çıkıntı yapmasını sağlamak için farenin başını ve boynunu yavaşça tutun.Gözün köşesi boyunca 100 mikrolitre floresan izleyici çözeltisi karışımı içeren bir insülin şırıngasını göze dik açıyla yerleştirin. Enjeksiyondan sonra, fareyi kartonun üzerine, uzuvları ve başı bantlanmış olarak sırtüstü pozisyonda hızlı bir şekilde yerleştirin. Submandibuler bez veya SMG izolasyonu için, stereoskopik mikroskop altında, SMG'lerin her iki tarafını da açığa çıkarmak için boynun epidermisini genel doku makası ile kesin.Daha sonra, bez dokusuna, kan damarlarına ve glandüler yapıya zarar vermeden kapsülü bez yüzeyinden nazikçe ayırmak için künt bir doku ayırma iğnesi kullanın. Özelleştirilmiş tutucuyu negatif basınç cihazına bağlayın ve negatif basınç etkisinin önceden uygun şekilde elde edilip edilmediğini kontrol edin. Açıkta kalan SMG'yi özelleştirilmiş tutucuya yavaşça yerleştirin ve solunum ve diğer koşulların neden olduğu hareket artefaktlarını azaltmak için negatif basınçlı emme yoluyla, fare gövdesinden mümkün olduğunca uzağa negatif basınçlı emme ile dokuyu emer.Bezin yeri seçildikten kısa bir süre sonra görüntülemeye başlayın. Z ekseni boyunca sıralı görüntüler elde etmek, üç boyutlu bir yapı oluşturmak için bezin yüzeyinden 70 ila 140 mikrona kadar dokuya iki mikron adım attı. Daha sonra, SMG'de vasküler geçirgenliği ölçmek için damarların hızlandırılmış görüntülemesini edinin.Explorer iletişim kutusunda, Yeni Klasör Ekle'ye tıklayın ve dosya adını değiştirin. Ardından, edinme sütununa geri dönün. XY'de, format 512 x 512'dir ve hız 400 hertz'dir.Çift yönlü X" i açın, yakınlaştırma için yakınlaştırma faktörünü 0,75 ve 1,5 olarak ayarlayın. Ardından, hızlandırılmış görüntüleme elde etmek için "Edinme Modu" altında XYT'yi seçin. Süreyi deneysel ihtiyaca göre 20 saniye, 30 dakika veya daha uzun olarak ayarlayın.Koşulları ayarladıktan sonra, fotoğraf oluşturan çerçevedeki iki kanalın ve üst üste binen kanalların vasküler görüntülemesini gözlemlemek ve ilgi alanlarını seçmek için "Canlı" ya tıklayın. Görüntülemeye başlamak için Başlat'a tıklayın. In vivo vasküler geçirgenlik testi ve farelerde kan damarlarının üç boyutlu görüntüleri.Submandibuler bezler burada gösterilmiştir. Kontrol grubunda, her iki izleyici de SMG'nin kan damarlarında mevcuttu. Küçük moleküler ağırlığı nedeniyle, FD4 kan damarlarından acini ve kanalların bazal taraflarına sızabildi, böylece acini ve kanalların şeklini açıkça tasvir etti.RD70, büyük boyutlu kan damarlarına ve mikro damarlara dağıtıldı. Kanal ligasyon grubunda, hem FD4 hem de RD70, aksinin bazal taraflarına ekstravaze edildi, bu da kanal ligasyonunun endotel bariyer fonksiyonunu bozabileceğini ve büyük moleküllere geçirgenliği artırabileceğini gösterdi. Ayrıca, yarı kantitatif FD4 ve RD70 floresan yoğunluğu sonuçları bu gözlemleri doğruladı.Üç boyutlu görüntüler, ligasyon grubundaki kan damarları etrafında FD4 ve RD70'in kontrol grubuna kıyasla çok daha belirsiz floresansını gösterdi. Dikkatli SMG izolasyonu ve uygun yerleştirme ve mikroskop, başarılı bir tespit için temel ön koşullardır. İşlemleri takiben, kırmızı kan hücrelerinin hareketi evlenerek kan akış hızı hesaplanabilir ve floresan etiketli kan hücrelerinin infiltrasyonu kovalanabilir.

in-vivo-vascular-permeability-detection-in-mouse-submandibular-gland

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JoVE Journal

Biology

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Tek hücreli fare Koni fotoreseptörlerin Emiş Kayıtlar

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Biyoloji

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Fare Tükürük Bezi Kök Hücre İzolasyonu

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Fare Modeller Meme Bezi Geliştirme ve Fonksiyon Değerlendirme

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Mitokondri İzole Fare Heart Geçirgenliği Geçiş Gözenek Açma Multi-parametre ölçümü

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Fare meme bezi Lokalize İlaç Teslim Intradüktal Enjeksiyon

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Ozmotik Su Geçirgenliği Katsayısı (P Ölçme<sub&gt; F</sub&gt;) Küresel Hücreleri: Bir Örnek olarak İzole Bitki protoplastlan

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

sertliği ölçme<em&gt; Ex Vivo</emAtomik Kuvvet Mikroskobu Kullanma&gt; Fare aorta

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

Vivo Algılama ve Rb Protein SUMOylation insan hücrelerinde analizi

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.