Name: observation of the ciliary movement of choroid plexus epithelial cells ex vivo
Uploaded: 2015-07-13T15:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo at JoVE.com

This work was supported by a Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (T.I.) and Grants-in-Aid for Scientific Research (C) from MEXT (S.T. and K.N).

protokoller ve Deney hayvanlarının kullanımı Yamanashi ve Waseda Üniversitesi Üniversitesi&#39;nde kurumsal hayvan bakımı ve kullanımı komiteleri tarafından onaylandı. Hayvan bakımı, kurumsal prensiplere uygun olarak gerçekleştirildi. 1. CPEC Hazırlık <ol><li> Aşağıdaki cihazlar ve malzemeleri hazırlayın: alttan aydınlatma iletilmesi tercihen bir stereo mikroskop; saatçi forseps bir çift (Dumont # 3 ya da # 4), alev sterilize düz işletim makas, alev sterilize; 20 ml buz gibi soğuk Leibovitz L-15 ihtiva eden ortam, iki steril 10 cm&#39;lik plastik tabaklar; 2 ml RT Leibovitz L-15 orta içeren 35 mm cam alt yemekler; % 70 etanol içeren 100 ml&#39;lik bir beher; yenidoğan fare yavrular. </li><li> Kısaca% 70 etanol içinde bir yenidoğan fare batırmak ve işletim makas kullanarak kafaları kesilmek suretiyle hızla euthanize. </li><li> Steril 10 cm di buz Leibovitz L-15 orta derhal baş koyunsh. </li><li> Beyin maruz kafatası açmak kesip, saatçi forseps çifti kullanarak calvaria cildi çıkarın ve ardından tüm beyni izole etmek için kranial sinirler kesilir. </li><li> Buz soğukluğunda Leibovitz L-15 ortamı içeren yeni bir çanak beyin aktarın ve beyin tam ortam içinde daldırılır emin, stereo bir mikroskop altında dikkate alınmalıdır. </li><li> Yukarı bakacak şekilde beynin dorsal yönünü ayarlayın ve koku ampuller (sağ-elli kişilik) 03:00 konumundaki ikamet böylece beyin yönlendirmek. Sol elinde forseps ile yavaşça beyin tutun. </li><li> İnce diseksiyon forseps (Dumont # 3 ya da # 4) sağ elinde kullanarak, beyin uzunlamasına fissür boyunca, serebral hemisfer bağlayan korpus kallosum ve parankiminde altında kesti. </li><li> Nazikçe yan taraflarına serebral hemisferlerin itin ve enine serebral fissür maruz kalmaktadır. </li><li> Th dışarı kısma hemisferlerin ayırınhemisfer ve talamus arasındaki e parankim. </li><li> Yavaşça lamina affixa ile hipokampus yan tarafına bağlı olduğu yanal ventriküler koroid pleksus çekin. </li><li> Taze Leibovitz L-15 orta içeren 35 mm cam alt çanak izole koroid pleksus aktarın ve ağırlığı (Malzeme Listesi) yavaşça yerine doku tutun kaplaması. </li></ol> CPEC Cilia 2. Canlı Görüntüleme <ol><li> 400 nm ve daha uzun 700 nm ve bu nötr yoğunluk (ND) filtreleri (% 25 ve% 6) daha kısa ışık ışık yolu eklenir engellemek için uygun ultraviyole (UV) ve anlaşıldığı (IR) kesim filtresi (ler) Onay ters mikroskop. </li><li> Kabaca gözle objektif lens odağını ayarlayın ve merkez ve odak Köhler aydınlatma uyacak, böylece daha sonra yoğuşturucuyu ayarlayın. Uygun bir diferansiyel girişim kontrast kaydedin (DIC) prizma, li bir DIC elemanı, hem de analiz ve polarize elemanlarght yolu DIC optik uymak. </li><li> Doku yüzeyinin yapısı en tanınabilir şekilde DIC prizma pozisyonu ile görüş kontrastını ayarlayın. Hedef hücrelerin her kirpikler hareketli iseniz, hareketli kirpikler net bir görünüm nedeniyle hareket gözle elde edilemez. </li><li> Video kamera ışık yolu değiştirin ve ışık gücünü artırmak için ND filtreleri kaldırabilirsiniz. </li><li> Görünümü ve odak alanını ayarlamak için mod odaklama kamerayı kullanın. Hangi video görüntüleri monitörde gerçek zamanlı olarak gösterilir, odaklama sırasında, hareketli kirpikler net bir görünüm mevcut değildir. </li><li> İstenilen süre (dakika saniye) 0,1 msn bir pozlama süresi ile 200 Hz kamerayı kullanın. Görüntü yığınının kazanılmasından sonra, tek kare net siliyer yapıları gösterecektir. Siliyer kenarları bulanık ise, kare hızını artırmak ya da daha kısa bir pozlama süresi kullanın. </li><li> Leibovitz içinde ötenazi sonra 25-60 dakika içinde CPEC kirpikler hareketini kaydedinL-15 ortamı. </li></ol> Silier Hareketinin Analizi 3. <ol><li> El ile bilgisayar monitöründe her cilium kalıplarını yenerek izleyebilirsiniz. Her cilium yörünge bilgisi için monte edilen fare işaretçisi, her karede Mark silier ucu pozisyonları. Ya daha fazla analiz için diğer daha genel uygulamalara aynı yazılımı veya ihracat kullanarak yörünge bilgileri analiz. Bu analiz, aşama verimi Tartışma tarif edilmektedir. </li><li> Gözle hareket iki mod, geri ve ileri-veya dönme içine yörüngeleri sınıflandırır. </li><li> Aşağıdaki formülü kullanarak siliyer dayak frekansı (CBF) hesaplayın: [CBF = (saniyedeki kare sayısı) / (Tek beat için kare ortalama sayısı)], bir siliyer ucu hareket şemasında elde edilebilir 14, (Şekil 3 ). Aynı hücrede başka kirpikler, her ciliu hareketi engelleyebilir, çünkü, birden fazla siliyer çırpma döngüsü için bu hesaplama tekrarlayınm, düzensizlik ile sonuçlanır. </li><li> Tek bir hücrenin içinde eksenleri yenerek ciliary açısal tekdüzelik analiz etmek, her yörünge (Şekil 4) için dayak açısı θ tanımlar. Geri ve ileri-yörüngeleri için, düz bir çizgi kirpikler ucu pozisyonlarını uygun ve satır x -Axis ile yapar açı olarak θ tanımlar. Rotasyonel yörüngeleri, bir elips pozisyonları uygun ve elipsin büyük eksen x -Axis ile yapar açı olarak θ tanımlar. Fitingin ayrıntıları Örnek Sonuçlar bölümünde tarif edilmektedir. </li><li> Her yörüngenin kantitatif açıklaması için, genelleştirilmiş boy oranı AR hesaplayın. Kısaca, göre yörünge döndürme - θ ve X boyunca dağılımının genişliğine arasındaki oran olarak AR tanımlar - ve y -axes (Şekil 4B). Detaylar Temsilcisi Sonuçlar bölümünde gösterilen ve yorumlama vardır, Önemi ve parametre sınırlama Tartışma açıklanmıştır. </li></ol> SEM 4. Numune Hazırlama Not: SEM kapsamlı bir şekilde CPECs üzerinde kirpikler durumunu değerlendirmek için önemli bir yöntemdir. SEM için örnekler hazırlamak için, standart bir prosedür daha önce 15 hafif değişiklikler kullanılmaktadır bildirdi. <ol><li> Beyin dokusu kesme önce, bir polietilen bir kap ile 5 ml&#39;lik bir cam şişe içinde fiksatif hazırlar. sabitleyici% 2 paraformaldehid, 0.1 M fosfat tampon maddesi, pH 7.4 içinde% 2.5 glutaraldehid (yarım Karnovsky çözeltisi 16) oluşur. </li><li> 1. adımda anlatıldığı gibi beyin doku parçalara ayır. </li><li> Kısaca, yeni bir tabağına Hank dengeli tuz çözeltisi (HBSS) ile izole edilmiş doku yıkayın ve daha sonra oda sıcaklığında 1 saat boyunca bir cam şişe içinde sabitleyici doku tamir. Tek kullanımlık transferi pipetler kullanın ve örnekleri ge koluntly. HBSS içinde durulama sonra, doku yapışkan hale gelir. <ol><li> Sabitleyici çözeltisi içine örnekleri aktarmak için yavaş bir damlacık olarak transfer pipeti doku ihtiva eden bir çözelti, az miktarda dışarı atılması ve sabitleştirici ekleyin. </li></ol></li><li> Tespit edildikten sonra, Fiksatif atmak ve fosfat tamponu ile üç kez doku durulayın. </li><li> Kalan aldehit yıkamak için% 10 sukroz çözeltisi doku daldırın. Aldehitlerin tamamen ortadan kaldırılmasını sağlamak 10 dakika boyunca çözelti içinde numune sokmak ve daha sonra, taze,% 10 sakaroz ile iki kez tekrarlayın. Bu adım, daha sonraki kademelerde, uygun post-fiksasyon elde etmek için önemlidir. </li><li> 30 dakika boyunca fosfat tamponu içinde% 1 ozmiyum tetroksit çözeltisi içinde doku daldırın ve sonrası sabitleme için buz üzerine yerleştirin. Numune rengine göre osmification derecesini Yargıç: aldehitler tamamen kaldırıldığında, örnek siyahtır. </li><li> Çift damıtılmış ile yoğun sonradan tespit doku örnekleri yıkayınd suyu birkaç kez. </li><li> Kademeli derişimlerde etanol içine daldırma ile örnekleri kurutmak, genellikle,% 65,% 75,% 85,% 95,% 99 ve% 100, 10 dakika her biri için. Yeni satın alınan şişe% 99.5 etanol içine moleküler elekler koyarak susuz etanol elde edilir. Susuz etanol ile üç kez dehidratasyon tekrarlayın. </li><li> 10 dakika boyunca, izoamil asetat, kritik nokta kurutma için bir ikame reaktifine susuz numuneler. İki kez bu adımı yineleyin. Bu reaktif hızla buharlaşır ve örnek yüzey gerilimi tarafından imha sonuçlanan kuru olabilir. Bu nedenle, tamamen kurumasını örnek yok. </li><li> Izoamil asetat nihai değişimi hemen sonra, çözücünün büyük bir kısmının çıkarılması, alüminyum folyo ile açık cam şişe kaydırmak ve kuru buz üzerinde şişe yerleştirin. Sıvı karbon dioksit, kritik nokta kurutma şişenin içine kolay akar, böylece bir iğne ya da ince forseps kullanılarak, şişenin ağzını kapsayan folyo çok delik açmak. Sonraki adıma geçinolabildiğince çabuk. </li><li> Bu adımda, kurutma odasına isoamil asetat taşınmasını en aza indirmek, ancak numune tamamen kritik nokta kurutmadan önce kurumaya izin vermeyin. Buna ek olarak, şişe üzerindeki don oluşumunu önlemek için gereğinden uzun bir süre boyunca kuru buz üzerinde örnek bırakmaz. </li><li> Çıkarmadan su dokusunda bulunan iken doku yüzey yapısı bozulmadan kalır sağlayan kritik nokta kurutucu içine doku örnekleri içeren folyo sarılı cam şişeleri aktarın. Kritik nokta kurutma işletim ile ilgili detaylı bilgiler üreticinin talimatlarına elde edilebilir. </li><li> Dikkatle mekanik zararı en aza indirmek için bir kürdan kullanarak örnekleri taşıyınız. Elde edilen kurutulmuş doku örnekleri kırılgandır. Bir iyon püskürtmeye kullanarak altın paladyum metal taslakları ve ceket örnekleri monte edin. </li></ol> SEM ile 5. Gözlem <ol><li> SEM ile gözlemleyin ve bir dijital kamera ile görüntüleri kaydedebilirsiniztaramalı elektron mikroskobu ile donatılmış. </li><li> Analiz için bir bilgisayara dijital görüntü verilerini aktarın. </li></ol>

<ol>
	<li>Nonami, Y., Narita, K., Nakamura, H., Inoue, T., Takeda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+ciliary+motility+on+choroid+plexus+epithelial+cells+during+the+perinatal+period.">Developmental changes in ciliary motility on choroid plexus epithelial cells during the perinatal period.</a> Cytoskeleton. 70 (12), 797-803 (2013).</li><li>Bisgrove, B. W., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+cilia+in+developmental+disorders+and+disease.">The roles of cilia in developmental disorders and disease.</a> Development. 133 (21), 4131-4143 (2006).</li><li>Fliegauf, M., Benzing, T., Omran, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+cilia+go+bad:+cilia+defects+and+ciliopathies.">When cilia go bad: cilia defects and ciliopathies.</a> Nature Reviews. Molecular Cell Biology. 8 (11), 880-893 (2007).</li><li>Oh, E. C., Katsanis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia+in+vertebrate+development+and+disease.">Cilia in vertebrate development and disease.</a> Development. 139 (3), 443-448 (2012).</li><li>Shah, A. S., Ben-Shahar, Y., Moninger, T. O., Kline, J. N., Welsh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motile+cilia+of+human+airway+epithelia+are+chemosensory.">Motile cilia of human airway epithelia are chemosensory.</a> Science(New York, N.Y). 325 (5944), 1131-1134 (2009).</li><li>Kiprilov, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells+in+culture+possess+primary+cilia+with+hedgehog+signaling+machinery).">Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery).</a> The Journal of Cell Biology. 180 (5), 897-904 (2008).</li><li>Hirokawa, N., Tanaka, Y., Okada, Y., Takeda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+flow+and+the+generation+of+left-right+asymmetry.">Nodal flow and the generation of left-right asymmetry.</a> Cell. 125 (1), 33-45 (2006).</li><li>Narita, K., Kozuka-Hata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+multiple+primary+cilia+reveals+a+novel+mode+of+ciliary+development+in+mammals.">Proteomic analysis of multiple primary cilia reveals a novel mode of ciliary development in mammals.</a> Biology Open. 1 (8), 815-825 (2012).</li><li>Takeda, S., Narita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+vertebrate+cilia,+towards+a+new+taxonomy.">Structure and function of vertebrate cilia, towards a new taxonomy.</a> Differentiation; Research in Biological Diversity. 83 (2), S4-S11 (2012).</li><li>Ikegami, K., Sato, S., Nakamura, K., Ostrowski, L. E., Setou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin+polyglutamylation+is+essential+for+airway+ciliary+function+through+the+regulation+of+beating+asymmetry.">Tubulin polyglutamylation is essential for airway ciliary function through the regulation of beating asymmetry.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (23), 10490-10495 (2010).</li><li>Foster, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+ciliary/flagellar+beating+of+Chlamydomonas.">Analysis of the ciliary/flagellar beating of Chlamydomonas.</a> Methods in Cell Biology. 91, 173-239 (2009).</li><li>Lechtreck, K. -. F., Sanderson, M. J., Witman, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+digital+imaging+of+ependymal+cilia+in+the+murine+brain.">High-speed digital imaging of ependymal cilia in the murine brain.</a> Methods in Cell Biology. 91, 255-264 (2009).</li><li>Okada, Y., Hirokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+nodal+cilia+movement+and+measurement+of+nodal+flow.">Observation of nodal cilia movement and measurement of nodal flow.</a> Methods in Cell Biology. 91, 265-285 (2009).</li><li>Chilvers, M. A., Rutman, A., O&#8217;Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+beat+pattern+is+associated+with+specific+ultrastructural+defects+in+primary+ciliary+dyskinesia.">Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia.</a> The Journal of Allergy and Clinical Immunology. 112 (3), 518-524 (2003).</li><li>Takeda, 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=Left-right+asymmetry+and+kinesin+superfamily+protein+KIF3A:+new+insights+in+determination+of+laterality+and+mesoderm+induction+by+kif3A-/-+mice+analysis.">Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/- mice analysis.</a> The Journal of Cell Biology. 145 (4), 825-836 (1999).</li><li>Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+formaldehyde-glutaraldehyde+fixative+of+high+osmolarity+for+use+in+electron+microscopy.">A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy.</a> The Journal of Cell Biology. 27, 137-138A (1965).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarını olduğunu beyan ederim. Bu yazıda izole koroid pleksus dokularda kirpikler hareketliliğini gözlemlemek için detaylı metodoloji raporlama amaçlamaktadır. Bilimsel yenilikler önceki çalışmalarda 1,8 olarak bildirilmiştir.

Bu yöntemin Perspektifler Burada açıklanan teknik daha önce yayınlanmış yöntemlere göre silya daha ayrıntılı bir analiz sağlamak olmamasına rağmen, bu tekniğin önemi kolayca siliyer hareketlilik ex vivo her türlü tarama uygulanabilir sistem ve maliyet etkinliği, sadeliği bulunur. Özellikle, TI Workbench gözlemlemek ve daha kolay siliyer hareketliliğini analiz araştırmacıları sağlayan basit ve kullanıcı dostu bir arayüz sağlar. Etkili otomatik izleme ...

Cilia are hair-like projections on the surface of most vertebrate cells, which have attracted attention by medical researchers because of a class of diseases termed ciliopathies2&#8211;4. Despite the ubiquitous expression of the organelle, a wide variety of ciliary functions have been reported, including motility and biosensing. For example, motile cilia on the mucoepithelial surface transport mucus5 and epithelial debris to the outlet of tracts, thereby preventing disease by clearing the surface of epithelia. Moreover, during early developmental periods and embryonic stages, cilia regulate the proliferation of stem cells6, and are involved in the determination of left&#8211;right asymmetry of the vertebrate body7. 
Choroid plexus epithelial cells (CPECs) are derivatives of neuroepithelial cells that cover the surface of the choroid plexus tissue in the brain, which play important roles in maintaining homeostasis of the intracranial environment by production of cerebrospinal fluid (CSF). It has been previously demonstrated that CPECs have multiple non-motile cilia that regulate the production of CSF through G-protein-coupled receptors that are specifically concentrated on the cilia8. Although these cilia had been regarded as quiescent non-motile cilia, it was discovered that some CPEC cilia exhibit transient motility during the neonatal period1. This finding was quite important because it revealed that so-called non-motile cilia are not necessarily immotile from the beginning of development and might display transient motility during specific time windows, possibly in response to specific physiological demands and functions9. To precisely describe the motile nature of CPEC cilia, it is necessary to develop an ex vivo observation system that encompasses analysis of the kinetic profiles unique to CPEC cilia. 
With respect to motility, although several technical reports have described observations of the motile cilia of the tracheal epithelium5,10, motile single-cell flagella11, so-called conventional motile cilia12, and nodal cilia13, detailed analytical methods applicable to relatively undulated structures such as the choroid plexus have not been well documented so far. Moreover, a high time resolution is required to analyze the ciliary movement of CPECs, in which expensive high-speed cameras are indispensable. To circumvent this necessity and simplify monitoring the ciliary motility of various cell types, a low cost, high-speed camera has been introduced, and an easily accessible and convenient method to record the motility of motile cilia, especially to describe the speed and pattern of motion of each cilium, has been developed1. Moreover, original image analysis software &#8220;TI Workbench&#8221; has been used here to facilitate detailed analysis of motility. Collectively, this method provides a new concise strategy to analyze ciliary motion together with correlative scanning electron microscopy (SEM), which can be adopted in a wide range of cilium research.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Required reagents&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>ethanol</td><td>Wako Chemicals</td><td>057-00456</td><td></td></tr><tr><td>Leibovitz L-15 medium</td><td>Life Technologies</td><td>11415-064</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for SEM preparation&lt;/strong&gt;</td></tr><tr><td>ethanol</td><td>Wako Chemicals</td><td>057-00456</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>Life Technologies</td><td>14170112</td><td></td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>Merck</td><td>1040051000</td><td></td></tr><tr><td>glutaraldehyde&amp;nbsp;</td><td>Nacalai tesque</td><td>17003-05</td><td></td></tr><tr><td>isoamyl acetate</td><td>Nacalai tesque</td><td>02710-95</td><td></td></tr><tr><td>Molecular Sieves 4A 1/8&amp;nbsp;</td><td>Wako Chemicals</td><td>130-08655</td><td>for preparation of anhydrous ethanol</td></tr><tr><td>phosphate buffer saline (PBS)</td><td>Sigma-Aldrich</td><td>D1408</td><td></td></tr><tr><td>phosphate buffer, 0.1 M</td><td></td><td></td><td>To make 100 ml, mix 19.0 ml of 0.1&amp;nbsp;M NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; and 81.0 ml of 0.1&amp;nbsp;M Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>monosodium phosphate (dihydrate)</td><td>Nacalai tesque</td><td>31718-15</td><td></td></tr><tr><td>disodium phosphate (anhydrous)</td><td>Nacalai tesque</td><td>31801-05</td><td></td></tr><tr><td>suclose</td><td>Nacalai tesque</td><td>30406-25</td><td></td></tr><tr><td>osmium tetroxide</td><td>Nisshin EM</td><td>300</td><td></td></tr><tr><td>dry ice</td><td></td><td></td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Tools and materials for dissection&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for both live imaging and SEM preparation&lt;/strong&gt;</td></tr><tr><td>stereo microscope</td><td>Olympus</td><td>SZX7</td><td></td></tr><tr><td>flat paper towel</td><td></td><td></td><td></td></tr><tr><td>&amp;Phi;10 cm plastic dish</td><td></td><td></td><td></td></tr><tr><td>100 ml beaker</td><td></td><td></td><td></td></tr><tr><td>straight operating scissors</td><td>Sansyo</td><td>S-2B</td><td></td></tr><tr><td>watchmaker forceps</td><td>Dumont</td><td>No.DU-3 or -4, INOX</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>glass bottom dish</td><td>Matsunami Glass</td><td>D110300</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for SEM preparation&lt;/strong&gt;</td></tr><tr><td>alminum foil</td><td></td><td></td><td></td></tr><tr><td>5 ml glass vial with a polyethylene cap</td><td>Nichiden Rika-Glass</td><td>PS-5A</td><td></td></tr><tr><td>transfer pipette</td><td>Samco Scientific</td><td>SM251-1S</td><td>for specimen tranfer</td></tr><tr><td>toothpick</td><td></td><td></td><td>for specimen transfer</td></tr><tr><td>ion sputter with gold-palladium</td><td>Hitachi</td><td>E-1030</td><td></td></tr><tr><td>critical point dryer</td><td>Hitachi</td><td>HCP-2</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Microscopic equipment and materials&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>100 W mercury lump housing and power supply</td><td>Olympus</td><td>U-ULH and BH2-RFL-T3</td><td></td></tr><tr><td>100 W mercury lamp</td><td>Ushio</td><td>USH103D</td><td></td></tr><tr><td>DIC condenser, n.a. 0.55</td><td>Olympus</td><td>IX-LWUCD</td><td></td></tr><tr><td>electrrical shutter</td><td>Vincent Associates</td><td>VS35S22M1R3-24 and VMM-D1</td><td>manual shutter can be used.</td></tr><tr><td>band-pass filter (400-700&amp;nbsp;nm, &amp;Phi;45&amp;nbsp;mm)</td><td>Koshin Kagaku</td><td>C10-110621-1</td><td></td></tr><tr><td>ND filter (&amp;Phi;45 mm)</td><td>Olympus</td><td>45ND6, 45ND25</td><td>combination of 25% and 6% ND filters are used</td></tr><tr><td>objective lens (water immersion) with DIC element</td><td>Olympus</td><td>UApo 40XW/340, n.a., 1.15 with IX-DPAO40</td><td></td></tr><tr><td>high-speed video camera</td><td>Allied Vision Technologies</td><td>GE680</td><td>&amp;#8805;200 Hz frame rate and 1 msec expose time</td></tr><tr><td>image acquisition / analysis software</td><td>in-hous software</td><td>TI Workbench</td><td>capable of acquisition at high frame rates.</td></tr><tr><td>PC for camera control / analysis</td><td>Apple</td><td>Mac Pro</td><td></td></tr><tr><td>vibration isolation table</td><td>Meiritsu Seiki</td><td>AD0806</td><td></td></tr><tr><td>weight for tissue</td><td>Warner Instruments</td><td>slice anchor kits</td><td>It can be made with nylon mesh glued to a U-shape squashed &amp;Phi;0.5&amp;nbsp;mm platinum wire.</td></tr><tr><td>cover glass (alternative weight for tissue)</td><td>Matsunami</td><td>made-to-order</td><td>A cover glass can be used as a tissue weight.</td></tr><tr><td colspan="4">&lt;strong&gt;for SEM&lt;/strong&gt;</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>scanning electron microscope</td><td>JEOL</td><td>JSM-6510</td><td></td></tr></tbody></table>

observation of the ciliary movement of choroid plexus epithelial cells ex vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Iş akışının bir bakış cihazları görüntü dahil olmak üzere Şekil 1 &#39;de gösterilmiştir. CPECs Canlı hareket gözlemleri Film 1 Bir perinatal fare izole CPECs bir film gösterir ve Film 2 Film 1 görüntüleri genişletilmiş bir görünümünü gösterir. Bireysel siliyer ipuçları filmlerde kıyasla hareketsiz görüntülerde daha az net olduğunu belirtmek gerekir. 2 gösteri...

Watch this Scientific Journal Video about Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo at JoVE.com

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Koroid Pleksus Epitel Hücreleri Silier Hareketi Gözlem<em&gt; Ex Vivo</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

observation-ciliary-movement-choroid-plexus-epithelial-cells-ex

Research

JoVE Journal

Neuroscience

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

12.1K Görüntüleme.  Waseda University. In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Video: Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Bir Ex Vivo modeli yaşlı insan Bruch'ın membran bir retina Pigment epitel hücrelerinin kültürü

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

Gelişim Biyolojisi

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Fare Beyin lateral ventrikül Ependimal Cilia Canlı Görüntüleme

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Nörobilim

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

Kemirgen Havayolu Epiteli içinde Cilia Motilite Yüksek Çözünürlüklü Görüntüleme için ex vivo Yöntemi

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Biyoloji

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Chick Ciliary Ganglion Nöronların İzolasyon ve Kültür

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Kemirgenlerde Cerrahi Olarak İndüklenen Glokom için Limbal Vasküler Pleksusun Tam Daire Koterizasyonu

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

Fare Koroid Pleksusunun Mikrodiseksiyonu ve Tüm Montajlı Taramalı Elektron Mikroskobu Görselleştirmesi

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Oküler Mikrovasküler Anjiyogenezin Ex Vivo Koroid Filizlenme Satomu

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Choroid Sprouting Assay to Study Ocular Microvascular Proliferation Ex Vivo

Source: Tomita, Y., et al. <a href="https://app.jove.com/v/61677">An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis.</a> J. Vis. Exp. (2020)
This video demonstrates the development of an ex vivo model of choroidal sprouting to study ocular microvascular angiogenesis. The assay involves culturing mouse eye explants containing the choroid tissue attached to the sclera and retinal pigment epithelium under specific conditions that promote choroidal microvascular sprouting.

Oküler mikrovasküler proliferasyonu ex vivo incelemek için koroid filizlenme testi

Source: Tomita, Y., et al. An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis. J. Vis. Exp. (2020)
This video demonstrates the ...

JoVE Encyclopedia of Experiments

Biyolojik Teknikler

Tahlil Teknikleri

Hücre bazlı analizler

Isolating Immune Cells from Mouse Choroid Plexuses

Source: Dominguez-Belloso, et al. <a href="https://app.jove.com/v/63487">Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure of isolating immune cells from mouse choroid plexuses. This procedure is used in neuroimmunology research to understand how immune cells in the choroid plexuses impact brain health and various neurological diseases.

Fare Koroid Pleksuslarından İmmün Hücrelerin İzole Edilmesi

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. ...

Koroid Pleksus Epitel Hücreleri Silier Hareketi Gözlem

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler