The authors would like to thank the Biomarker Imaging&#160;Research Laboratory (BIRL) at Sunnybrook Research Institute for their histology services. Support for this work was provided by the Terry Fox Foundation, the Canadian Breast Cancer Foundation&#8208;the Prairie&#8208;NWT as well as a CIHR grant, #MOP-97996.

1. Spécimen <ol><li> Exciser chirurgicalement les glandes mammaires de type sauvage CDH1 ainsi que des souris IGFBP7 nulle ou trois jours après le début de la lactation. </li><li> Répartir les glandes sur des lames de verre pour aider à retrouver natif glande mammaire morphologie. </li></ol> 2. Fixation et Traitement des tissus <ol><li> Fixer les glandes mammaires chez neutre tamponné 4% PFA O / N à 4 ° C. </li><li> Conserver les glandes éthanol à 70% avant le traitement des tissus. </li><li> Transférer les glandes de petites cassettes de traitement des tissus. </li><li> Traiter les tissus à l&#39;aide d&#39;un processeur de tissus automatisé <ol><li> Déshydrater les tissus dans l&#39;augmentation de l&#39;éthanol et des bains de xylène de 70% d&#39;éthanol pendant 45 minutes, deux fois dans l&#39;éthanol 95% pendant 45 minutes, 3 fois dans 100% d&#39;éthanol pendant 1 heure et 2 heures dans du xylène pendant 45 min. </li><li> Imprégner les tissus de paraffine 3 fois pendant 1 heure chaque dans le vide avec une pression appliquée. </li></ol></li><li> Incluez les tissus en paraffine pour former des blocs, Pour la coupe. </li></ol> 3. Histologie et d&#39;îlot Imaging <ol><li> Couper les blocs de paraffine en utilisant un microtome rotatif jusqu&#39;à ce que l&#39;excès de paraffine est retirée. </li><li> Utiliser une fraiseuse verticale pour percer des trous de 1 mm dans au moins deux coins du bloc de paraffine perpendiculaire à la cassette. </li><li> Monter le bloc de tissu sur le microtome rotatif. </li><li> Mettre en place le système d&#39;imagerie de côté d&#39;îlot 19 à l&#39;avant du microtome. </li><li> Image optique de côté d&#39;îlot capturer avant la coupe. </li><li> Couper des rubans de quatre sections à 5 um d&#39;épaisseur sur le microtome. <ol><li> Transférer les rubans au bain d&#39;eau froide. </li><li> Séparer les deuxième et quatrième sections de ruban et les monter sur des lames de microscope. Choisir les deuxième et quatrième sections fournit un écart de 5 um entre les sections. </li><li> Développez chaque section dans un bain d&#39;eau chaude (48 o C) à dérider, puis re-monter sur la lame de microscope. REMARQUE: CUtting, de montage, unwrinkling les sections provoquent des distorsions sur la section, telles que la déchirure, pli, le retrait et l&#39;expansion. Ces artefacts compliquent l&#39;enregistrement des sections histologiques. </li><li> Colorer les sections avec H &amp; E en utilisant une coloration automatique. </li><li> Lamelle les diapositives à l&#39;aide d&#39;une colleuse automatique. </li><li> Numériser les diapositives à l&#39;aide d&#39;un scanner de diapositives de l&#39;histologie numérique à la résolution d&#39;intérêt. Pour ce protocole le grossissement est de 20x et la résolution est de 0,47 um. </li><li> Bas-échantillonner les images d&#39;histologie à la résolution des images du côté d&#39;îlot, 18 um. </li></ol></li></ol> 4. L&#39;image Enregistrement <ol><li> Segmentation de l&#39;image et de la sélection du point <ol><li> En images côté d&#39;îlot de mesurer les valeurs de pixel des trous d&#39;enregistrement et d&#39;utiliser la valeur moyenne comme seuil fixe pour segmenter les trous d&#39;enregistrement dans les coins du bloc de paraffine. </li><li> Depuis quelques pièces supplémentaires pourraient aussi être segmentés parutilisant le seuil fixé, utiliser la circularité et la zone des objets segmentés pour trouver les trous et jeter les objets supplémentaires. Pour ce faire, écrire un petit code et trouver le rapport de (4π x surface) / (périmètre) 2 pour les objets segmentés. Ce ratio pour les objets ronds est 1. </li><li> Pour chaque glande mammaire, sélectionner une image de côté d&#39;îlot comme référence et aligner le reste des images du côté d&#39;îlot à la référence à l&#39;aide du centre des trous d&#39;enregistrement et de techniques d&#39;enregistrement basé repère. </li><li> Pour les images alignées du côté d&#39;îlot, un segment ou extrait du tissu de l&#39;arrière-plan manuellement. Utilisez l&#39;objet le plus importante dans le masque pour le reste du protocole. </li><li> Pour H &amp; E sections suivez les étapes ci-dessous pour la segmentation automatique. <ol><li> Utilisez Otsu technique de seuillage 20 images de segments de l&#39;arrière-plan et créer des masques binaires des images d&#39;histologie. </li><li> Identifier et sélectionner l&#39;objet le plus importante dans chaque masque en utilisant la histogram des objets marqués. </li><li> Extraire les un pixel de large points limites de deux histologie et masques de côté d&#39;îlot. </li><li> Utilisez le code de la chaîne algorithme 21, pour représenter les points de la frontière par une séquence de linéaire par morceaux s&#39;adapte. </li></ol></li></ol></li><li> Inscription rigide initiale <ol><li> Utiliser les descripteurs de Fourier algorithme 22, pour trouver la première transformation rigide entre les points limites de l&#39;histologie et de leurs images correspondantes du côté d&#39;îlot. Cette première transformation comprend les facteurs traduction, rotation et échelle. </li><li> Transformer chaque image avec l&#39;histologie initiale transformée obtenue à partir de l&#39;étape précédente. </li></ol></li><li> Raffinement de l&#39;enregistrement rigide <ol><li> Retirer les sections de pointe de haute courbure du contour de l&#39;histologie en utilisant un filtre de roulement à billes 23. </li><li> Sélectionnez 500 points des points limites d&#39;histologie restantes à l&#39;aide de façon aléatoire de distribution uniforme. </li><li> Transformer le lahistologie points limites aléatoires avec la transformation initiale obtenue à partir de descripteurs de Fourier. </li><li> Sélectionner l&#39;ensemble des points de la frontière du côté d&#39;îlot et utiliser itératives points les plus proches (ICP) de l&#39;algorithme 24 pour trouver la transformation rigide entre les points limites de côté d&#39;îlot, de la destination et histologie points limites aléatoires. </li><li> Transformer les images histologiques alignées obtenues à partir de l&#39;étape précédente et de la pile d&#39;images histologiques alignés crée le volume de l&#39;histologie. </li><li> Utiliser un logiciel de visualisation 3D pour créer une image visuelle du volume de l&#39;histologie. </li></ol></li><li> Affichage de la pile des images au grossissement de 5x <ol><li> Bas-échantillonner les images d&#39;histologie originaux 5x grossissement. </li><li> Recadrage de la région d&#39;intérêt dans l&#39;une des images d&#39;histologie. </li><li> Calculer l&#39;emplacement de cette région dans les autres images histologiques 5x utilisant la combinaison des transformations rigides provenant des deux étapes d&#39;enregistrement. </li><li> Recadrer la régions d&#39;intérêt à la même région de la taille de toutes les autres images d&#39;histologie. </li><li> Enfin affiner l&#39;alignement entre les régions manuellement. Écrire un programme qui se superpose deux images et permet de sélectionner les valeurs de rotation et de translation de l&#39;une des images sur l&#39;autre, puis enregistre l&#39;image transformée lorsque l&#39;alignement est acceptée. </li><li> Voir les piles des régions d&#39;histologie 5x alignés en utilisant un logiciel de visualisation 3D. </li></ol></li></ol>

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The authors have nothing to disclose

Dans cette étude, nous avons développé un enregistrement flux de travail d&#39;image à reconstruire un volume d&#39;histologie 3D de série d&#39;images d&#39;histologie 2D, qui ne nécessitent pas de points de repère internes choisis au hasard ou marqueurs fiduciaires implantés dans le tissu, ce qui pourrait fausser le tissu. Par le procédé décrit, des images du côté d&#39;îlot optiques elles-mêmes sont utilisées comme images de référence avant la coupe. Nous utilisons des trous percés dans extérieure...

IGFBP7 (insuline like growth factor protéine de liaison 7) est un membre de la famille des protéines IGF-contraignant, et a été montré pour se lier au récepteur de l&#39;IGF1 4. Régulation à la baisse de IGFBP7 est connu pour être en corrélation avec un mauvais pronostic dans le cancer du sein 5, tandis que la réintroduction de IGFBP7 dans des modèles de xénogreffe de tumeur inhibe fortement la croissance des tumeurs par le biais 6 induction de l&#39;apoptose et la sénescence cellulaire 7. Afin d&#39;étudier les effets de IGFPB7, une souris IGFBP7 nul a été créé 5 (données non publiées). Bien que ces souris ne développent pas de tumeurs, ils montrent des changements dans l&#39;histologie de l&#39;ovaire, du muscle et du foie, ainsi que des défauts dans la structuration du développement des glandes mammaires (données non publiées). Le phénotype défectueux a été indiqué que les souris nulles ont des portées moins nombreuses et sont incapables de soutenir plusieurs grandes portées (données non publiées). Volumes d&#39;histologie 3D ont le potentiel de fournir des informat utileion pour les analyses et l&#39;évaluation quantitative et comparative des résultats pathologiques dans les images médicales volumétriques. Confocale tridimensionnelle, la microscopie à deux photons peut fournir à haute résolution cellule information morphologique de la glande à mesure locale 14, mais il a un champ de vision limité et la profondeur. reconstruction du volume d&#39;histologie fournit plus d&#39;informations sur une plus grande étendue spatiale. En utilisant des approches traditionnelles de la distorsion est prévu au cours de la préparation de coupes histologiques, tels que le retrait, l&#39;expansion, de larmes et de plis. Ces déformations, il est difficile d&#39;enregistrer des images histologiques en série dans une pile 3D à reconstruire un volume 3D. Comme le nombre de sections consécutives avec des défauts augmente les similitudes entre les sections intactes est réduite et rend le processus d&#39;enregistrement plus compliqué, par conséquent. Différentes méthodes ont été proposées pour enregistrer les coupes histologiques et à créer un vo histologie continuelume. Certaines techniques dépendent de la variation d&#39;intensité et 8, et d&#39;autres sont basées sur la forme des sections 9. Pour certains spécimens les structures anatomiques peuvent être utilisés comme points de repère 10,11 ainsi que les méthodes d&#39;enregistrement basé repère-12,13. Mais ces structures internes peuvent ne pas être détectable dans tout le volume et pour certains spécimens pas fiables structures anatomiques peuvent être identifiés. Certains groupes ont utilisé une approche d&#39;inscription par paire et enregistré des images d&#39;histologie consécutives un à l&#39;autre à l&#39;aide des contours ou des structures anatomiques 16-18. Enregistrement des coupes en série d&#39;histologie à l&#39;autre sans l&#39;utilisation d&#39;images de référence peut se propager erreur d&#39;enregistrement et modifier la forme réelle du volume de l&#39;histologie. Approche d&#39;inscription par paire repose sur la cohérence de la forme des coupes histologiques et les structures internes à travers l&#39;empilement des images; par conséquent, il nécessite échantillonnage dense de l&#39;échantillon, quipeut-être pas toujours possible, par exemple, pour les échantillons cliniques. Dans ce pipeline, nous utilisons des images du côté d&#39;îlot comme un ensemble d&#39;images de référence pour la reconstruction du volume de l&#39;histologie 19. Images du côté d&#39;îlot sont prises des blocs de tissus de paraffine après montage sur le microtome et avant chaque section est coupée. Ainsi, les dommages à l&#39;individu des coupes en série coupe n&#39;interfère pas avec l&#39;enregistrement des coupes en série 8,11,15. Nous capturons les images du côté d&#39;îlot d&#39;une manière différente des autres groupes. Les images de visage de bloc optique sont obtenues par une lentille télécentrique pour éliminer ou minimiser le canon et la distorsion de perspective, ce qui se produit généralement lors de l&#39;utilisation des lentilles de l&#39;optique réguliers. C&#39;est l&#39;un des avantages de l&#39;approche proposée par rapport aux autres méthodes publiées, qui effectuent l&#39;imagerie de côté d&#39;îlot à l&#39;aide de lentilles ordinaires. Les images sont prises avec un léger angle oblique à utiliser la réflexion de la surface du bloc pour un rehaussement du contraste entre le tisssurface ue et de la paraffine et pour éliminer l&#39;ombre du tissu en profondeur, sous la surface de la paraffine. Un filtre photographique est également utilisée pour polariser la lumière provenant de la surface du bloc et le tissu pour équilibrer le contraste 19. Afin de corriger le déplacement du bloc sur le microtome rotatif, de deux à trois trous sont percés dans les coins du bloc, qui sont facilement détectables dans les images du côté d&#39;îlot. Les centres de gravité de ces trous sont utilisés avec inscription rigide à base de point de repère pour aligner les images du côté d&#39;îlot.

<table border="0" cellpadding="0" cellspacing="0" width="852">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">16% PFA</td>
			<td style="width:179px;">VWR International</td>
			<td style="width:119px;">15710</td>
			<td style="width:295px;">16% Paraformaldehyde solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Small tissue processing cassettes</td>
			<td style="width:179px;">VWR International</td>
			<td>CA95029-956</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Leica ASP300 Automated Tissue processor</td>
			<td style="width:179px;">Leica</td>
			<td>14047643515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">100% ethanol</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S25307B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Xylene</td>
			<td>VWR International&#160;</td>
			<td>CA95057-822</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Paraffin&#160;</td>
			<td style="width:179px;">Thermo Fisher</td>
			<td style="width:119px;">39501006</td>
			<td>Paraplast Tissue Embedding Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica EG 1160 Embedding Centre</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica rotary microtome</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Milling machine</td>
			<td style="width:179px;">Argo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Microscope slides</td>
			<td style="width:179px;">VWR International&#160;</td>
			<td>CA48312-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">H&#38;E stain</td>
			<td style="width:179px;">VWR International</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Automatic stainer</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Coverslips&#160;</td>
			<td style="width:179px;">VWR International&#160;</td>
			<td>48404-452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">MEDITE RCM 7000 Glass Coverslipper</td>
			<td style="width:179px;">MEDITE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica SCN400 slide scanner</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">MATLAB</td>
			<td style="width:179px;">MathWorks Inc</td>
			<td>MATLAB 2007b</td>
			<td>Development software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">MeVisLab</td>
			<td style="width:179px;">MeVis Medical Solutions AG</td>
			<td>MeVisLab 2.1</td>
			<td>3D visualization software</td>
		</tr>
	</tbody>
</table>

reconstruction of 3-dimensional histology volume and its application to study mouse mammary glands

reconstruction du volume d&#39;histologie facilite l&#39;étude de la forme 3D et le changement de volume d&#39;un organe au niveau des macrostructures constituées de cellules. Il peut également être utilisé pour étudier et valider de nouvelles techniques et des algorithmes d&#39;imagerie médicale volumétrique et thérapies. Création d&#39;atlas 3D à haute résolution des différents organes 1,2,3 est une autre application de la reconstruction du volume de l&#39;histologie. Ceci fournit une ressource pour l&#39;enquête structures tissulaires et la relation spatiale entre les différentes fonctions cellulaires. Nous présentons une approche d&#39;enregistrement d&#39;image pour la reconstruction du volume de l&#39;histologie, qui utilise un ensemble d&#39;images de côté d&#39;îlot optiques. Le volume de l&#39;histologie reconstruite représente une forme fiable de l&#39;échantillon traitées sans erreur propagée d&#39;inscription post-traitement. Les hématoxyline et éosine (H &amp; E) sections colorées de deux glandes mammaires de souris ont été enregistrés à leurs images correspondantes du côté d&#39;îlot en utilisant des points limites extraites de l&#39;éditionges de l&#39;échantillon en histologie et du côté d&#39;îlot images. La précision de l&#39;enregistrement a été évalué visuellement. L&#39;alignement des macrostructures des glandes mammaires a également été évaluée visuellement à haute résolution. Cette étude définit les différentes étapes de ce pipeline d&#39;enregistrement d&#39;image, allant de l&#39;excision de la glande mammaire par l&#39;intermédiaire de la reconstruction 3D du volume de l&#39;histologie. Bien 2D images histologiques révèlent les différences structurelles entre les paires de sections, le volume de l&#39;histologie 3D permet de visualiser les différences de forme et de volume des glandes mammaires.

Un écueil de techniques de microscopie traditionnels est que la compréhension d&#39;un organe à l&#39;échelle microscopique est limitée à un champ de vision à la fois. Même &quot;divulgation total&quot; diapositives, qui fournissent des sections entières de diapositives, ne parviennent pas à fournir des informations en trois dimensions. Avec le développement de la coulisse ensemble, les technologies de numérisation dynamiques, notre capacité à voir un article dans sa totalité a augmenté, mais l&#39;extra...

Watch this Scientific Journal Video about Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands at JoVE.com

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Reconstruction de 3-Dimensional Volume histologique et son application à l&#39;étude de souris glandes mammaires

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

reconstruction-3-dimensional-histology-volume-its-application-to

Research

JoVE Journal

Biology

14.3K vues.  University of Toronto. We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands. 

Vidéo: Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Le barreau d&#39;échelle Marche Tâche: Un système de notation et son application pratique.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Objectif transplantation chez le poisson zèbre et son application dans l&#39;analyse de mutants Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Souris cellules épithéliales mammaires forme Mammospheres cours de la différenciation lactogène

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

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

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

Evaluation de développement des glandes mammaires et fonction dans des modèles murins

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

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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

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

Injection intra-canalaire pour localisée Drug Delivery de la glande mammaire de souris

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

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

Un Exonuclease analyse basée sur la fluorescence pour caractériser DmWRNexo, orthologue humain progéroïde WRN Exonuclease, et son application à d&#39;autres nucléases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

À haut débit d&#39;analyse de l&#39;image de Sphéroïdes tumorales: Une application logicielle conviviale pour mesurer la taille de Sphéroïdes automatiquement et avec précision

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Utilisation de la souris des cellules tumorales mammaires pour enseigner Biologie Concepts de base: un module de laboratoire simple

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Mesure de boucles de volume de pression chez la souris

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...