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).

Les protocoles et l&#39;utilisation des animaux de laboratoire ont été approuvés par les soins des animaux et de l&#39;utilisation des comités institutionnels de l&#39;Université de Yamanashi et de l&#39;Université de Waseda. soins aux animaux a été effectuée conformément aux directives institutionnelles. 1. Préparation CPEC <ol><li> Préparer des appareils et des matériaux suivants: un microscope stéréo, de préférence capable de transmettre l&#39;illumination du fond; une paire de pinces horloger (Dumont N ° 3 ou n ° 4), la flamme stérilisé, des ciseaux d&#39;exploitation droites, flamme stérilisé; deux plats 10 cm en plastique stériles contenant 20 ml glacée moyen Leibovitz L-15; Plats à fond de verre de 35 mm contenant 2 ml RT Leibovitz milieu L-15; un bêcher de 100 ml contenant 70% d&#39;éthanol; souriceaux nouveau-nés. </li><li> Immerger brièvement une souris néonatale dans 70% d&#39;éthanol et euthanasier rapidement par décapitation en utilisant les ciseaux d&#39;exploitation. </li><li> Placez la tête immédiatement Leibovitz milieu L-15 glacée dans le stérile à 10 cm dish. </li><li> Retirer la peau des calvaria utilisant la paire de forceps horloger, ouvrir le crâne pour exposer le cerveau, et puis couper les nerfs crâniens pour isoler l&#39;ensemble du cerveau. </li><li> Transférer le cerveau à un nouveau plat contenant Leibovitz milieu L-15 glacée et observer au microscope stéréo, faire en sorte que le cerveau est complètement immergé dans le milieu. </li><li> Réglez la face dorsale du cerveau vers le haut, et d&#39;orienter le cerveau de sorte que les bulbes olfactifs résident à la position trois heures (pour les droitiers). Maintenez le cerveau en douceur avec la pince à la main gauche. </li><li> En utilisant les pinces à dissection fines (Dumont N ° 3 ou # 4) dans la main droite, couper le corps calleux et sous le parenchyme reliant les hémisphères cérébraux, le long de la fissure longitudinale du cerveau. </li><li> Poussez doucement les hémisphères cérébraux loin aux côtés latéraux et d&#39;exposer la fissure transversale cérébrale. </li><li> Séparer les hémisphères par pincement sur the parenchyme entre l&#39;hémisphère et le thalamus. </li><li> Tirez doucement sur le plexus choroïde ventriculaires latérales qui est attaché à la face latérale de l&#39;hippocampe par le affixa lamina. </li><li> Transférer les plexus choroïde isolés au plat à fond de verre de 35 mm contenant du milieu frais Leibovitz L-15, et superposer un poids (liste des matières) doucement pour maintenir le tissu en place. </li></ol> 2. imagerie en direct de CPEC Cilia <ol><li> Confirmez ultraviolet bon (UV) et le filtre (s) déduit (IR) de coupe pour bloquer la lumière inférieure à 400 nm et plus de 700 nm, et que la densité neutre (ND) filtres (25% et 6%) sont insérés dans le trajet de la lumière du microscope inversé. </li><li> Régler la netteté de l&#39;objectif plus ou moins à l&#39;oeil, et ensuite ajuster le condenseur afin que le centre et le foyer pour se conformer à l&#39;éclairage de Köhler. Insérer un contraste interférentiel différentiel approprié (DIC) prisme, un élément DIC, ainsi que des éléments de l&#39;analyseur et le polariseur dans le lichemin lutte pour se conformer à l&#39;optique DIC. </li><li> Régler le contraste de vue par la position du prisme DIC sorte que la structure de la surface du tissu est plus reconnaissable. Si tous les cils des cellules cibles sont mobiles, une vision claire des cils mobiles ne peut pas être obtenue par l&#39;œil en raison de leur mouvement. </li><li> Modifiez le chemin de lumière à la caméra vidéo, et de supprimer les filtres ND pour augmenter la puissance lumineuse. </li><li> Utilisez l&#39;appareil photo en mode de mise au point de régler le champ de vision et de concentration. Au cours de mise au point, dans lequel les images vidéo sont affichées en temps réel sur l&#39;écran, une vue claire des cils mobiles ne sont pas disponibles. </li><li> Utilisez l&#39;appareil photo à 200 Hz avec un temps d&#39;exposition de 0,1 ms pour la période souhaitée (secondes à quelques minutes). Après l&#39;acquisition de la pile de l&#39;image, des images uniques seront afficher structures ciliaires claires. Si bords ciliaires sont floues, d&#39;augmenter le taux de trame ou utiliser un temps d&#39;exposition plus courte. </li><li> Enregistrer le mouvement de CPEC cils au sein 25-60 minutes après l&#39;euthanasie en LeibovitzMilieu L-15. </li></ol> 3. Analyse du mouvement ciliaire <ol><li> Suivre manuellement battant motifs de chaque cil sur le moniteur de l&#39;ordinateur. Marquer les positions de pointe ciliaire dans chaque image avec le pointeur de la souris, qui sont assemblés pour obtenir des informations trajectoire de chaque cil. Soit analyser les informations trajectoire en utilisant le même logiciel ou l&#39;exportation vers d&#39;autres applications plus générales pour une analyse plus approfondie. L&#39;efficacité de cette étape d&#39;analyse est décrite dans la discussion. </li><li> Classer les trajectoires en deux modes de mouvement, de va-et-vient ou de rotation, par oeil. </li><li> Calculer la fréquence ciliaire de battage (CBF) en utilisant la formule suivante: [CBF = (nombre d&#39;images par seconde) / (nombre moyen d&#39;images pour un seul battement)] 14, qui peut être obtenu à partir d&#39;un schéma pointe ciliaire de mouvement (Figure 3 ). Répéter ce calcul pour de multiples cycles de battement ciliaire, parce que d&#39;autres cils sur la même cellule peut interférer avec le mouvement de chaque cilium, ce qui entraîne l&#39;irrégularité. </li><li> Pour analyser l&#39;uniformité angulaire du battement ciliaire axes dans une seule cellule, définir le θ de l&#39;angle de battement pour chaque trajectoire (Figure 4). Pour trajectoires de va-et-vient, adapter les postes de la pointe des cils à une ligne droite, et de définir l&#39;angle θ que la ligne fait avec axe x. Pour trajectoires de rotation, adapter les postes à une ellipse, et de définir l&#39;angle θ que l&#39;axe majeur de l&#39;ellipse fait avec axe x. Détails de la ferrure sont décrits dans la section des résultats représentatifs. </li><li> Pour une description quantitative de chaque trajectoire, calculer généralisée rapport d&#39;aspect AR. En bref, la trajectoire de rotation - θ et AR définir comme étant le rapport entre la largeur de la distribution le long de x - y et -axes (figure 4B). Les détails sont affichés dans la section des résultats représentatifs, et l&#39;interprétation, L&#39;importance et la limitation du paramètre sont décrites dans la discussion. </li></ol> 4. Préparation d&#39;échantillons pour MEB Remarque: SEM est une méthode importante pour évaluer l&#39;état des cils sur CPEC d&#39;une manière globale. Pour préparer des échantillons pour SEM, une procédure standard précédemment rapporté 15 est employé avec de légères modifications. <ol><li> Avant la dissection du tissu du cerveau, préparer le fixateur dans un flacon en verre de 5 ml avec un bouchon en polyéthylène. Le fixateur est constitué de 2% de paraformaldehyde, 2,5% de glutaraldéhyde (moitié de la solution de 16 Karnovsky) dans 0,1 M de tampon phosphate, pH 7,4. </li><li> Disséquer les tissus du cerveau, comme décrit dans l&#39;étape 1. </li><li> Rincer brièvement le tissu isolé avec une solution saline équilibrée de Hank (HBSS) dans un nouveau plat, puis fixer le tissu dans le fixateur dans le flacon de verre pendant 1 heure à température ambiante. Utilisez pipettes de transfert unique et manipuler les spécimens gently. Après rinçage dans HBSS, le tissu devient collant. <ol><li> Pour transférer les échantillons dans la solution de fixateur, lentement expulser une petite quantité de solution contenant le tissu de la pipette de transfert en gouttelettes et ajouter au fixateur. </li></ol></li><li> Après fixation, jeter le fixateur et rincer le tissu avec tampon phosphate trois fois. </li><li> Plonger le tissu dans une solution de saccharose à 10% pour se laver les aldéhydes restants. Pour assurer l&#39;élimination complète des aldéhydes, plonger les échantillons dans la solution pendant 10 min, puis répéter deux fois avec des produits frais 10% de saccharose. Cette étape est importante pour parvenir à post-fixation correcte dans les étapes suivantes. </li><li> Plonger le tissu dans une solution de 1% de tétroxyde d&#39;osmium dans un tampon phosphate pendant 30 min, puis placer sur la glace pour la post-fixation. Juger du degré de osmification par la couleur de l&#39;échantillon: quand aldéhydes sont complètement enlevés, l&#39;échantillon est noir. </li><li> Laver les échantillons de tissus postdéterminés abondamment avec double distilléed l&#39;eau plusieurs fois. </li><li> Déshydrater les échantillons par immersion dans des concentrations échelonnées d&#39;éthanol, généralement 65%, 75%, 85%, 95%, 99% et 100%, pendant 10 minutes chaque. Obtenir de l&#39;éthanol anhydre en plaçant des tamis moléculaires dans 99,5% d&#39;éthanol à partir d&#39;une bouteille nouvellement acheté. Répétez avec déshydratation de l&#39;éthanol anhydre à trois reprises. </li><li> Placer les échantillons déshydratés dans de l&#39;acétate d&#39;isoamyle, un réactif de substitution pour un séchage au point critique, pendant 10 min. Répéter deux fois cette étape. Ce réactif évapore rapidement et l&#39;échantillon peut devenir sèche, provoquant la destruction par la tension de surface. Par conséquent, ne pas sécher l&#39;échantillon complètement. </li><li> Après le dernier échange de l&#39;acétate d&#39;isoamyle, enlever la plupart du solvant, immédiatement envelopper le flacon en verre ouvert avec du papier d&#39;aluminium et placez le flacon sur glace sèche. En utilisant une aiguille ou une pince fine, faire plusieurs trous dans la feuille de recouvrement de l&#39;embouchure de la fiole, de telle sorte que le dioxyde de carbone liquide coule facilement dans la fiole dans le sécheur à point critique. Passez à l&#39;étape suivanteaussi vite que possible. </li><li> Dans cette étape, minimiser le report de l&#39;acétate d&#39;isoamyle dans la chambre de la sécheuse, mais ne laissez pas l&#39;échantillon sécher complètement avant séchage au point critique. En outre, ne pas laisser l&#39;échantillon sur de la glace sèche pour une inutilement beaucoup de temps pour éviter la formation de givre sur le flacon. </li><li> Transférer les flacons de verre papillote contenant les échantillons de tissus dans le sèche-linge au point critique qui assure la structure de la surface du tissu reste intact tandis que l&#39;eau contenue dans l&#39;élimination du tissu. Des informations détaillées sur le fonctionnement du sèche point critique peut être obtenu à partir des instructions du fabricant. </li><li> Manipulez les échantillons soigneusement à l&#39;aide d&#39;un cure-dent pour minimiser les dommages mécaniques. Les échantillons de tissus séchées résultantes sont fragiles. Montez les échantillons sur les moignons métalliques et manteau avec de l&#39;or-palladium en utilisant une pulvérisation d&#39;ions. </li></ol> 5. observation par MEB <ol><li> Observez par SEM et enregistrer des images avec un appareil photo numériqueéquipé d&#39;un microscope électronique à balayage. </li><li> Transférer des données d&#39;image numériques à un ordinateur pour analyse. </li></ol>

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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>

Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrentes. Ce document vise à communiquer l&#39;méthodologie détaillée pour observer la motilité des cils dans les tissus du plexus choroïde isolés. Nouveautés scientifiques ont été rapportés dans les études antérieures 1,8.

Perspectives de cette méthode Bien que la technique décrite ici ne prévoit pas une analyse plus détaillée des cils que les méthodes déjà publiées, la signification de cette technique réside dans la simplicité du système et l&#39;efficacité des coûts, qui peut être facilement appliquée au dépistage tout type de motilité ciliaire ex vivo. En particulier, TI Workbench fournit une interface simple et conviviale qui permet aux chercheurs d&#39;observer et d&#39;analyser...

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.

Vue d&#39;ensemble du flux de production est représenté sur la Figure 1, y compris les images des dispositifs. Observations de mouvement direct de CPEC Film 1 montre un film de CPEC isolés à partir d&#39;une souris périnatale et Movie 2 montre une vue agrandie des images dans le film 1. Il convient de noter que des conseils individuels ciliaires sont moins claires dans les images fixes par r...

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.

Observation du Mouvement ciliaire des cellules épithéliales plexus choroïde<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 vues.  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.

Vidéo: 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 modèle organotypique cornéenne aiguë de l&#39;herpès simplex virus type épithéliales je 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 ...

Recherche

Neurosciences

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.

Une description visuelle de la dissection de la vascularisation cérébrale et de surface associées Méninges et le plexus choroïde du cerveau de rat

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 préparatifs de l&#39;ampoule Vomeronasal organes et accessoire olfactive Intact

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 Imagerie du calcium chez la souris Olive inférieure

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.

Préparation du nerf sciatique de rat pour la neurophysiologie ex vivo

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.

Cultures organotypiques du cortex humain adulte comme modèle ex vivo pour la transplantation et la validation de cellules souches humaines

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.

Microdissection et microscopie électronique à balayage à monture entière Visualisation du plexus choroïde de souris

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

Silençage génique sélectif dans le plexus choroïde

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.

Pleins feux sur l’auteur : Perspectives et innovations dans les techniques de manipulation de l’expression génique pour la recherche sur le plexus choroïde 

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

Knockdown ciblé des gènes du plexus choroïde

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