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).

Die Protokolle und die Verwendung von Versuchstieren wurden von den institutionellen Tierpflege und Verwendung Ausschüsse an der University of Yamanashi und Waseda University zugelassen. Tierpflege wurde in Übereinstimmung mit den Richtlinien des Instituts durchgeführt. 1. CPEC Vorbereitung <ol><li> Bereiten Sie die folgenden Vorrichtungen und Materialien: ein Stereo-Mikroskop, vorzugsweise geeignet zur Übertragung von Beleuchtung von unten; ein Paar von Uhrmacher Pinzette (Dumont # 3 oder # 4), flammsterilisiert, gerade Betriebs Schere, flamm sterilisiert; zwei sterile 10 cm Plastikschalen mit 20 ml eiskaltem Leibovitz L-15-Medium; 35 mm Glasbodenschalen mit 2 ml RT Leibovitz L-15-Medium; ein 100 ml Becherglas, das 70% Ethanol; neonatalen Maus Welpen. </li><li> Kurz eintauchen eine neonatale Maus in 70% Ethanol und schnell einschläfern durch Enthauptung mit dem Betriebs Schere. </li><li> Platzieren Sie den Kopf sofort in eiskaltem Leibovitz L-15-Medium in das sterile 10 cm dish. </li><li> Entfernen Sie die Haut von den Schädeldecke mit Hilfe der beiden Uhrmacher Zangen, schnitt den Schädel, um das Gehirn freizulegen, und dann schneiden Sie die Hirnnerven, die ganze Gehirn zu isolieren. </li><li> Übertragen Sie das Gehirn zu einem neuen Schale mit eiskaltem Leibovitz L-15-Medium und beobachten unter dem Stereomikroskop, um sicherzustellen, dass das Gehirn vollständig in das Medium eingetaucht. </li><li> Stellen Sie die Dorsalseite des Gehirns nach oben ein, und richten Sie das Gehirn, so dass die Riechkolben wohnen an der Drei-Uhr-Position (für Rechtshänder). Halten Sie das Gehirn vorsichtig mit der Pinzette in die linke Hand. </li><li> Mit den feinen Pinzette Dissektion (Dumont # 3 oder # 4) in der rechten Hand, schneiden Sie den Balken und unterhalb des Parenchyms Verbinden der Gehirnhälften, die entlang der Längsspalte des Großhirns. </li><li> Schieben Sie die Gehirnhälften entfernt an den Seiten und setzen die Querhirnspalte. </li><li> Trennen Sie die Hemisphären durch Kneifen out the Parenchym zwischen der Hemisphäre und Thalamus. </li><li> Ziehen Sie vorsichtig die seitlichen Ventrikel Plexus, die auf der lateralen Seite des Hippocampus durch die Lamina affixa angebracht ist. </li><li> Übertragen Sie die isolierten Plexus auf die 35 mm Glasbodenschale mit frischen Leibovitz L-15-Medium, und überlagern ein Gewicht (Materialliste) leicht, um das Gewebe in Position zu halten. </li></ol> 2. Live-Imaging von CPEC Cilia <ol><li> Überprüfen Sie die ordnungsgemäße ultraviolette (UV) und geschlossen (IR) Cut-Filter (n) zu sperren Licht kürzer als 400 nm und länger als 700 nm, und das Neutraldichtefilter (ND) (25% bzw. 6%) in den Strahlengang eingelegt des invertierten Mikroskops. </li><li> Stellen Sie den Fokus des Objektivs grob durch Auge und stellen Sie dann den Kondensator, so dass das Zentrum und Fokus auf die Köhler-Beleuchtung entsprechen. Legen Sie eine geeignete Differentialinterferenzkontrast (DIC) Prisma, ein DIC-Element sowie Analysator und Polarisator Elemente in der liGHT PATH um das DIC Optik entsprechen. </li><li> Stellen Sie den Kontrast der Blick von der DIC-Prisma Position, so dass die Struktur der Gewebeoberfläche ist besonders erkennbar. Wenn alle Zilien der Zielzellen sind beweglich, eine klare Sicht auf bewegliche Zilien kann nicht mit dem Auge wegen ihrer Bewegung erreicht werden. </li><li> Ändern Sie den Lichtweg zu der Videokamera und der ND-Filter zu entfernen, um die Lichtleistung zu erhöhen. </li><li> Verwenden Sie die Kamera in Fokusmodus, um das Sichtfeld und Fokus einzustellen. Während der Fokussierung, bei der Videobilder in Echtzeit auf dem Monitor angezeigt wird, ist eine klare Sicht auf bewegliche Zilien nicht verfügbar. </li><li> Mit der Kamera bei 200 Hz mit einer Belichtungszeit von 0,1 ms für den gewünschten Zeitraum (Sekunden bis Minuten). Nach der Übernahme des Bildstapels wird Einzelbilder klar ciliary Strukturen anzuzeigen. Wenn ciliary Kanten sind unscharf, erhöht die Bildrate oder verwenden Sie eine kürzere Belichtungszeit. </li><li> Nehmen Sie die Bewegung der Flimmerhärchen CPEC innerhalb 25-60 Minuten nach der Euthanasie in LeibovitzL-15-Medium. </li></ol> 3. Analyse der Flimmerbewegung <ol><li> Manuell verfolgen schlagen Muster jedes cilium auf dem Computermonitor. Filter ciliary Spitzenpositionen in jedem Rahmen mit dem Mauszeiger, die Flugbahn-Informationen jedes cilium zusammengesetzt werden. Entweder analysieren die Flugbahn Daten mit der gleichen Software oder den Export in andere allgemeinere Anwendungen für die weitere Analyse. Die Effizienz dieses Analyseschritt auch in der Diskussion beschrieben. </li><li> Klassifizierung der Bahnen in zwei Bewegungsarten, hin- und her bzw. Dreh, mit dem Auge. </li><li> Berechnen Sie die Zilienschlag Frequenz (CBF) mit der folgenden Formel: [CBF = (Anzahl der Bilder pro Sekunde) / (durchschnittliche Anzahl der Bilder für einen einzelnen Schlag)] 14, die aus einer ciliary Spitze Bewegungsdiagramm erhalten werden können (Abbildung 3 ). Wiederhole diese Berechnung für mehrere Zilienschlag Zyklen, weil andere Zilien auf der gleichen Zelle kann mit der Bewegung jedes ciliu interferierenm, was zu Unregelmäßigkeiten. </li><li> Um die Winkel Gleichmäßigkeit der Zilienschlag Achsen innerhalb einer einzelnen Zelle zu analysieren, definieren das Schlagen Winkel θ für jede Flugbahn (Abbildung 4). Für Hin-und-her-Trajektorien, passen die Positionen der Zilien Spitze auf eine gerade Linie, und definieren θ als Winkel der Linie macht mit x-Achse. Für Dreh Trajektorien, passen die Positionen zu einer Ellipse und definieren θ als Winkel der Hauptachse der Ellipse macht mit x-Achse. Details der Armatur sind im Repräsentative Ergebnisse beschrieben. </li><li> Zur quantitativen Beschreibung der einzelnen Flugbahn berechnen verallgemeinerte Seitenverhältnis AR. Kurz gesagt, dreht die Trajektorie durch - θ und AR definieren als das Verhältnis zwischen der Breite der Verteilung entlang x - und y-Achsen (4B). Einzelheiten sind in der Repräsentative Ergebnisse Schnitt gezeigt, und die Interpretation, Bedeutung, und die Begrenzung der Parameter sind in der Diskussion beschrieben. </li></ol> 4. Probenvorbereitung für die SEM Anmerkung: SEM ist ein wichtiges Verfahren, um den Status der Zilien auf CPECs in umfassender Weise auszuwerten. Um Proben für SEM vorzubereiten, berichtete ein Standardverfahren zuvor 15 mit geringen Modifikationen verwendet. <ol><li> Vor der Zerlegung der Gewebe aus dem Gehirn, bereiten Sie die Fixiermittel in einer 5 ml-Glasgefäß mit einer Polyethylenkappe. Das Fixiermittel besteht aus 2% Paraformaldehyd, 2,5% Glutaraldehyd (Halb Karnovsky-Lösung 16) in 0,1 M Phosphatpuffer, pH 7,4. </li><li> Präparieren Sie das Gewebe aus dem Gehirn, wie in Schritt 1 beschrieben. </li><li> Kurz abspülen isolierten Gewebes mit ausgewogener Hank-Salzlösung (HBSS) in ein neues Gericht und dann fix das Gewebe in der Fixiermittel in dem Glasfläschchen für 1 Stunde bei Raumtemperatur. Verwenden Sie Einweg-Transferpipetten und behandeln die Proben gently. Nach Spülen in HBSS, wird das Gewebe klebrig. <ol><li> Um die Proben in die Fixierungslösung zu übertragen, zu vertreiben langsam eine kleine Menge der Lösung, die das Gewebe von der Transferpipette als ein Tröpfchen und zu dem Fixiermittel. </li></ol></li><li> Nach der Fixierung, entsorgen Sie die Fixierungsmittel und spülen Sie das Gewebe mit Phosphatpuffer dreimal. </li><li> Tauchen Sie das Gewebe in einer 10% igen Saccharoselösung zum Auswaschen der verbleibenden Aldehyde. Um vollständige Beseitigung von Aldehyden zu gewährleisten, tauchen Sie die Proben in der Lösung für 10 Minuten und wiederholen Sie dann zweimal mit frischem 10% Saccharose. Dieser Schritt ist wichtig, die richtige Postfixierung in den nachfolgenden Schritten zu erreichen. </li><li> Tauchen des Gewebes in einer Lösung von 1% Osmiumtetroxid in Phosphatpuffer für 30 min und dann auf Eis für Postfixierung. Beurteilen Sie den Grad der osmification von der Probe Farbe: wenn Aldehyde vollständig entfernt ist die Probe schwarz. </li><li> Die post-fixierten Gewebeproben umfassend Waschen mit Doppel destilliertd Wasser mehrmals. </li><li> Dehydratisieren die Proben durch Eintauchen in abgestufte Konzentrationen von Ethanol, in der Regel 65%, 75%, 85%, 95%, 99% und 100%, für jeweils 10 min. Erhalten wasserfreiem Ethanol, indem Molekularsiebe in 99,5% Ethanol aus einer neu gekauften Flasche. Wiederholen Dehydratisierung mit wasserfreiem Ethanol dreimal. </li><li> Legen Sie die dehydrierten Proben in Isoamylacetat, einem Substitutions Reagenz zum Trocknen am kritischen Punkt, für 10 min. Wiederholen Sie diesen Schritt zweimal. Dieses Reagenz schnell verdampft und die Probe kann trocken werden, was zu einer Zerstörung durch die Oberflächenspannung. Daher nicht die Probe vollständig trocknen. </li><li> Nach dem letzten Austausch von Isoamylacetat, entfernen Sie das meiste Lösungsmittel, sofort wickeln Sie das offene Glas mit Aluminiumfolie, und legen Sie das Fläschchen auf Trockeneis. Mittels einer Nadel oder einer feinen Pinzette, stellen mehrere Löcher in die Folie für den Mund des Fläschchens, so daß flüssiges Kohlendioxid fließt leicht in die Phiole in dem kritischen Punkt Trockner. Fahren Sie mit dem nächsten Schrittso schnell wie möglich. </li><li> In diesem Schritt minimieren die Übertragung von Isoamylacetat in die Kammer des Trockners, aber lassen Sie die Probe vollständig austrocknen, bevor Trocknen am kritischen Punkt. Hinzu kommt, dass die Probe nicht auf Trockeneis verlassen unnötig lange Zeit, um die Bildung von Frost auf dem Fläschchen zu vermeiden. </li><li> Übertragen Sie die Folie eingeGlasFläschchen, die die Gewebeproben in den kritischen Punkt Trockner, dass die Oberflächenstruktur des Gewebes bleibt erhalten, während das Wasser im Gewebe enthaltenen gewährleistet. Detaillierte Informationen über den Betrieb der kritischen Punkt Trockner können aus den Anweisungen des Herstellers bezogen werden. </li><li> Behandeln Sie die Proben vorsichtig mit einem Zahnstocher, um mechanische Schäden zu minimieren. Das resultierende getrocknete Gewebeproben sind zerbrechlich. Montieren Sie die Proben auf Metall Stubs und Mantel mit Gold-Palladium unter Verwendung eines Ionen Sputter. </li></ol> 5. Beobachtung durch SEM <ol><li> Beobachten von SEM und Bilder aufnehmen mit einer Digitalkamerazu Rasterelektronenmikroskop ausgestattet. </li><li> Übertragen digitaler Bilddaten auf einen PC zur Auswertung. </li></ol>

<ol>
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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>

Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen. Dieses Papier zielt darauf ab, die Berichterstattung detaillierte Methodik, um die Beweglichkeit der Zilien in isolierten Plexus Gewebe zu beobachten. Scientific Neuheiten wurden in früheren Studien 1,8 berichtet.

Perspektiven dieser Methode Obwohl die hier beschriebene Technik ist eine detailliertere Analyse der Zilien als bisher veröffentlichten Verfahren liefern, die Bedeutung dieser Technik besteht in der Einfachheit des Systems und Kosteneffizienz, die leicht an jede Art von Screening Zilienmotilitätsstörungen ex vivo angewendet werden kann. Insbesondere stellt TI Workbench eine einfache und benutzerfreundliche Schnittstelle, die Forschern die Beobachtung und leichter analysieren Zili...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>for both live imaging and SEM preparation</td>
			<td></td>
			<td></td>
			<td></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>&#934;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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom dish</td>
			<td>Matsunami Glass</td>
			<td>D110300</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></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>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></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 nm, &#934;45 mm)</td>
			<td>Koshin Kagaku</td>
			<td>C10-110621-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ND filter (&#934;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>&#62;= 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 &#934;0.5mm platinum wire.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM</td>
			<td></td>
			<td></td>
			<td></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>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</td>
			<td>Wako Chemicals</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>1040051000</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</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&#160;</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 M NaH2PO4 and 81.0 ml of 0.1 M Na2HPO4&#160;</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 align="right">300</td>
			<td></td>
		</tr>
		<tr>
			<td>dry ice</td>
			<td></td>
			<td></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.

Ein Überblick über den Arbeitsablauf ist in Figur 1 dargestellt ist, einschließlich der Bilder der Geräte. Live Motion Beobachtungen CPECs Film 1 zeigt ein Film von CPECs aus einer perinatalen Maus isoliert und Movie 2 zeigt eine vergrößerte Ansicht der Bilder in Film 1. Es wird darauf hingewiesen, dass einzelne ciliary Tipps sind in Vergleich mit denen in Filmen Einzelbilder weniger klar w...

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.

Beobachtung der Flimmerbewegung von Plexus Epithelzellen<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

12.0K Aufrufe.  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.

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

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

Ex Vivo Organotypische Corneal Modell der akuten Epithelial Herpes Simplex Virus Typ I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

Forschung

Neurowissenschaften

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

Ein Visual Beschreibung der Dissektion der Hirnoberfläche Gefäßsystem und assoziierten Hirnhäute und der Aderhaut Plexus aus Rattenhirn

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex vivo Zubereitungen der Intact Vomeronasalorgan und akzessorischen olfaktorischen Bulbus

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Maus Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Vorbereitung des Rattenischiasnervs für die Ex-vivo-Neurophysiologie

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Organotypische Kulturen des adulten humanen Kortex als Ex-vivo-Modell für die Transplantation und Validierung humaner Stammzellen

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

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.

Mikrodissektion und Whole-Mount-Rasterelektronenmikroskopie Visualisierung des Aderhautplexus der Maus

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

Selektives Gen-Silencing im Plexus choroideus

Targeted Knockdown of Genes in the Choroid Plexus

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

Autor im Rampenlicht: Erkenntnisse und Innovationen bei Techniken zur Manipulation der Genexpression für die Erforschung des Plexus choroideus 

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.

Gezielter Knockdown von Genen im Plexus aderoid

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...