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. Proben <ol><li> Verbrauchsteuern die Brustdrüsen chirurgisch aus Wildtyp-CDH1 sowie IGFBP7-null-Mäusen 3 Tage nach dem Einsetzen der Laktation. </li><li> Verbreiten Sie die Drüsen auf Glasobjektträger zu helfen, wieder Muttermilchdrüsenmorphologie. </li></ol> 2. Fixierung und Gewebeverarbeitung <ol><li> Befestigen Sie die Milchdrüsen in neutral gepuffertem 4% PFA O / N bei 4 ° C. </li><li> Bewahren Sie die Drüsen in 70% Ethanol vor der Gewebeverarbeitung. </li><li> Übertragen Sie die Drüsen, kleine Gewebeverarbeitung Kassetten. </li><li> Verarbeiten Sie die Gewebe mit Hilfe eines automatisierten Gewebeprozessor <ol><li> Dehydratisierung der Gewebe in zunehmenden Ethanol und Xylol Bäder von 70% Ethanol für 45 min, 2 mal in 95% Ethanol für 45 Minuten, 3 mal in 100% Ethanol für 1 Stunde und 2 mal in Xylol für 45 min. </li><li> Dringen das Gewebe mit Paraffin 3 Mal für jeweils 1 h in einem Vakuum mit Druckvorgabe. </li></ol></li><li> Betten Sie das Gewebe in Paraffin zu Blöcken, Für das Schneiden. </li></ol> 3. Histologie und Blockface Imaging <ol><li> Schneiden Sie die Paraffinblöcke mit Hilfe eines Rotationsmikrotoms, bis das überschüssige Paraffin wird entfernt. </li><li> Verwenden eine Vertikalfräsmaschine 1 mm Bohrungen in mindestens zwei Ecken der Paraffinblock senkrecht zu der Kassette. </li><li> Montieren Sie den Gewebeblock auf der Rotationsmikrotoms. </li><li> Richten Sie die blockface Abbildungssystem 19 vor dem Mikrotom. </li><li> Optischen Bild blockface Erfassen vor dem Schneiden. </li><li> Schneiden Bänder aus vier Abschnitten bei 5 um Dicke am Mikrotom. <ol><li> Übertragen Sie die Bänder an den kalten Wasserbad. </li><li> Trennen Sie die zweiten und vierten Abschnitt des Bandes und montieren sie auf Objektträgern. Wahl der zweiten und vierten Abschnitt eine Lücke zwischen 5 &amp;mgr; m Abschnitte. </li><li> Erweitern Sie jeden Abschnitt in einem warmen Wasserbad (48 ° C), um sie unwrinkle, dann wieder montieren auf dem Objektträger. HINWEIS: CUtting, Montage, Glättungs die Abschnitte führen, dass einige Verzerrungen auf dem Abschnitt, wie Reiß, falten, Schrumpfung und Expansion. Diese Artefakte erschweren die Registrierung der Histologieschnitten. </li><li> Färben der Schnitte mit H &amp; E mit einem automatischen stainer. </li><li> Deckglas die Folien mit einem automatischen Eindeckautomat. </li><li> Digitalisieren Sie die Folien mit einem digitalen Histologie Dia-Scanner mit einer Auflösung von Interesse. Aus diesem Protokoll ist die Vergrößerung 20x und die Auflösung ist 0,47 um. </li><li> Down probieren Sie die Histologie Bilder an die Auflösung des blockface Bilder, 18 um. </li></ol></li></ol> 4. Bildregistrierung <ol><li> Bildsegmentierung und Punktauswahl <ol><li> In blockface Bilder messen die Pixelwerte der Ausrichtungslöcher und benutzen den Durchschnittswert als eine feste Schwelle zu segmentieren die Ausrichtungslöcher in den Ecken des Paraffinblocks. </li><li> Da einige Zusatzteile kann auch durch segmentiert werdenmit dem festgelegten Schwellenwert, verwenden Sie die Rundheit und den Bereich der segmentierten Objekte, um die Löcher zu finden und entsorgen Sie die zusätzlichen Objekten. Um dies zu tun, schreibe einen kleinen Code und finden Sie das Verhältnis von (4π x Fläche) / (Perimeter) 2 für die segmentierten Objekte. Dieses Verhältnis für runde Objekte 1 ist. </li><li> Für jede Brustdrüse, wählen Sie eine blockface Bild als Referenz, und richten Sie den Rest der blockface Bilder zum Referenz, indem die Mitte der Anmeldung Löcher und Wahrzeichen-basierte Registrierungstechniken. </li><li> Für die ausgerichteten blockface Bilder manuell Segment oder extrahieren Sie die Gewebe aus dem Hintergrund. Nutzen die meisten großen Objekt durch in der Maske für den Rest des Protokolls. </li><li> Für H &amp; E Abschnitte die folgenden Schritte für die automatische Segmentierung. <ol><li> Verwenden Otsu Schwellwerttechnik 20 bis Segment Bilder aus dem Hintergrund und erstellen binäre Masken der Histologie Bilder. </li><li> Identifizieren und wählen Sie die ansehnliche Objekt in jeder Maske mit dem histogram der markierten Objekte. </li><li> Extrahieren Sie die ein Pixel breite Grenzpunkte von beiden Histologie und blockface Masken. </li><li> Verwenden Chain-Code-Algorithmus 21, um die Grenzpunkte durch eine Folge von stückweise linearen darstellen passt. </li></ol></li></ol></li><li> Initial Starre Registrierung <ol><li> Verwenden Fourier Deskriptoren Algorithmus 22, zu finden, die erste starre Transformation zwischen den Grenzpunkten der Histologie und ihre entsprechenden blockface Bilder. Diese erste transformieren umfasst die Translation, Rotation und Skalierungsfaktoren. </li><li> Verwandeln Sie jedes Bild mit der Histologie aus dem vorherigen Schritt die Anfangs Transformation erhalten. </li></ol></li><li> Verfeinerung der Starre Registrierung <ol><li> Entfernen Sie die hohe Krümmung Randabschnitte von der Histologie Kontur mit einem rollenden Ball Filter 23. </li><li> Wählen Sie 500 Punkte aus den verbleibenden Histologie Grenzpunkte zufällig mit gleichmäßigen Verteilung. </li><li> Transformieren Sie die dieHistologie Zufallsgrenzpunkte mit der aus der Fourier-Deskriptoren erhalten anfänglichen Transformation. </li><li> Wählen Sie die ganze Reihe von blockface Grenzpunkte und benutzen Iterative Closest Point (ICP) Algorithmus 24, um die starre Transformation zwischen den blockface Grenzpunkte, Ziel und Histologie Zufallsgrenzpunkte zu finden. </li><li> Transformation der ausgerichteten Histologie Bilder aus dem vorhergehenden Schritt erhalten, und der Stapel ausgerichtet Histologie Bilder erstellt die Histologie Volumen. </li><li> Verwenden Sie ein 3D-Visualisierungs-Software, um eine visuelle Bild der Histologie Volume zu erstellen. </li></ol></li><li> Stack anzeigen der Bilder bei 5-facher Vergrößerung <ol><li> Down probieren Sie die Original Histologie Bilder, um die Vergrößerung 5x. </li><li> Crop die Region von Interesse in einer der Histologie Bilder. </li><li> Berechnen der Position dieser Region in anderen 5x Histologie Bilder unter Verwendung der Kombination der starren Transformationen aus den zwei Schritten der Registrierung. </li><li> Schneiden Sie das regions von Interesse für die gleichen Größenbereich in allen anderen histologischen Bilder. </li><li> Schließlich verfeinern die Ausrichtung zwischen den Regionen manuell. Ein Programm zu schreiben, die zwei Bilder überlagert, und ermöglicht die Auswahl der Werte für Rotation und Translation eines der Bilder gegenüber dem anderen ein und speichert dann das transformierte Bild, wenn die Ausrichtung angenommen wird. </li><li> Sehen Sie sich die Stapel der ausgerichteten 5x Histologie Regionen mit einem 3D-Visualisierungssoftware. </li></ol></li></ol>

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The authors have nothing to disclose

In dieser Studie haben wir eine Bildregistrierung Workflow, um ein 3D-Histologie Volumen aus 2D-Serien Histologie Bilder, die nicht internen zufällig ausgewählten Landmarken oder implantiert Bezugsmarker im Gewebe, die das Gewebe verzerren könnten erfordert rekonstruieren entwickelt. Durch das beschriebene Verfahren werden optische Bilder blockface sich als die Referenzbilder vor dem Schneiden verwendet. Wir verwenden externe Löcher in dem Paraffinblock gebohrt, um beim Ausrichten der Bilder blockface unterstützen ...

IGFBP7 (Insulin-like growth factor binding protein 7) ist ein Mitglied der IGF-bindende Proteinfamilie, und es wurde gezeigt, um die IGF-1-Rezeptor-4 zu binden. Herabregulierung von IGFBP7 ist bekannt, mit einer schlechten Prognose bei Brustkrebs 5 korreliert werden, während die Wiedereinführung von IGFBP7 in Xenograft-Tumormodellen hemmt stark die Tumorwachstums 6 durch Induktion von Apoptose und Zellalterung 7. Um die Auswirkungen der IGFPB7 zu untersuchen, wurde ein IGFBP7 null Maus 5 (unveröffentlichte Daten) erstellt. Während diese Mäuse keine Tumore entwickeln, Veränderungen in der Histologie des Eierstock-, Muskel-und Leber sowie Mängel in der Brustdrüse Entwicklungs Strukturierung (unveröffentlichte Daten) zeigen sie. Das defekte Phänotyp wurde angegeben als die null-Mäuse haben kleinere Wurfgrößen und sind unfähig, mehrere große Würfe (unveröffentlichte Daten) zu erhalten. 3D-Histologie Bände haben das Potenzial, um nützliche Informat bietenIonen für quantitative und vergleichende Analysen und Bewertung der pathologischen Befunde in Volumen medizinische Bilder. Dreidimensionales konfokales können zwei-Photonen-Mikroskopie hoher Auflösung Zelle morphologische Information der Drüse an lokalen Speicherbereich 14 zu schaffen, aber eine begrenzte Sichtfeld und Tiefe hat. Histologie Volumenrekonstruktion liefert mehr Informationen über einen viel größeren räumlichen Ausdehnung. Verwendung traditioneller Ansätze gewisse Verzerrung während der Herstellung histologischer Schnitte, wie z. B. Schrumpfung, Expansion, Risse und Falten erwartet. Diese Verzerrungen machen es schwierig, seriellen histologischen Bilder in ein 3D-Stapel Register, um ein 3D-Volumen zu rekonstruieren. Da die Anzahl von aufeinanderfolgenden Abschnitten mit Defekten erhöht die Ähnlichkeiten zwischen intakten Abschnitte reduziert und folglich ist der Registrierungsprozess komplizierter. Verschiedene Methoden wurden vorgeschlagen, um histologische Schnitte zu registrieren und eine kontinuierliche Histologie vo erstellenlume. Einige Techniken sind abhängig von Intensitätsvariationen 8, und andere werden von der Form der Abschnitte 9 basiert. Bei einigen Proben können die anatomischen Strukturen als Landmarken 10,11 zusammen mit Wahrzeichen-basierte Registrierungsverfahren 12,13 verwendet werden. Aber diese inneren Strukturen möglicherweise nicht nachweisbar sein in der gesamten Menge und für einige Proben keine zuverlässigen anatomischen Strukturen identifiziert werden können. Einige Gruppen haben eine paarweise Anmeldung Ansatz verwendet und registriert in Folge Histologie Bilder einem zum anderen mit Konturen oder anatomischen Strukturen 16-18. Registrieren von Serien Histologie Abschnitte miteinander, ohne die Verwendung von Referenzbildern kann die Registrierung Fehler zu propagieren und ändern Sie die tatsächliche Form der Histologie Lautstärke. Paarweise Anmeldung Ansatz beruht auf der Konsistenz der Form der Histologie Abschnitten und den inneren Strukturen im gesamten Stapel der Bilder; daher dichter Abtastung der Probe, es erfordert dienicht immer möglich ist, z. B. für klinische Proben. In dieser Pipeline verwenden wir blockface Bilder als eine Reihe von Referenzbildern für die Histologie 19 Volumenrekonstruktion. Blockface Bilder der Paraffingewebeblöcke nach der Montage am Mikrotom genommen und vor jeder Abschnitt wird abgeschnitten. Somit muss Beschädigungen einzelner Serienschnitte Schnitt nicht mit der Registrierung der Serienschnitte 8,11,15 stören. Wir erfassen die blockface Bilder in einer anderen Weise von den anderen Gruppen. Die optischen Block Gesichtsbilder werden von einem telezentrischen Objektiv zu beseitigen oder zu minimieren, den Lauf und perspektivische Verzerrung, die in der Regel tritt auf, wenn mit regulären Linsen in der Optik erhalten. Dies ist einer der Vorteile der vorgeschlagenen Vorgehensweise gegenüber den anderen Methoden veröffentlicht, die blockface Bildgebung durchführen mit regulären Linsen. Die Bilder werden in einem geringen spitzen Winkel getroffen werden, um die Reflexion von der Oberfläche des Blocks zur Kontrastverstärkung zwischen dem TISS verwendenue und Paraffinoberfläche und den Schatten des Gewebes in der Tiefe unter der Oberfläche zu beseitigen Paraffin. Photographische Filter wird auch verwendet, um das Licht, das von der Blockoberfläche und dem Gewebe, um den Kontrast 19 ausgleichen zu polarisieren. Um die Verschiebung des Blocks auf der Rotationsmikrotom korrigieren, zwei bis drei Löcher in den Ecken des Blocks, die in den blockface Bilder leicht nachweisbar sind gebohrt. Die Schwerpunkte dieser Löcher werden zusammen mit Wahrzeichen-basierten starren Anmeldung benutzt, um die Bilder blockface auszurichten.

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

Histologie Volumenrekonstruktion erleichtert die Untersuchung von 3D-Form-und Volumenänderung eines Organs auf der Ebene der Makrostrukturen aus Zellen aufgebaut. Es kann auch verwendet werden, um zu untersuchen und Validierung neuer Techniken und Algorithmen, die in volumetrischen medizinischen Bildgebung und Therapie werden. Erstellen von 3D-High-Resolution-Atlanten von verschiedenen Organen, 1,2,3 ist eine weitere Anwendung der Histologie Volumenrekonstruktion. Dies stellt eine Ressource für die Untersuchung von Gewebestrukturen und die räumliche Beziehung zwischen verschiedenen zellulären Funktionen. Wir präsentieren eine Bildregistrierung Ansatz für die Histologie Volumenrekonstruktion, die eine Reihe von optischen blockface Bilder verwendet. Das rekonstruierte Histologie Volumen stellt eine zuverlässige Form der verarbeiteten Probe ohne breiteten Post-Processing-Registrierungsfehler. Die Hämatoxylin und Eosin (H &amp; E) gefärbten Schnitten von zwei Maus-Brustdrüsen wurden zu den entsprechenden Bildern mit blockface Grenzpunkte von der ed extrahiert registriertges der Probe in der Histologie und blockface Bilder. Die Genauigkeit der Registrierung wurde visuell bewertet. Die Ausrichtung der Makrostrukturen der Brustdrüsen wurde visuell mit hoher Auflösung untersucht. Diese Studie umreißt die verschiedenen Schritte des Bildregistrierungs Pipeline, von Entfernung der Brustdrüse durch den 3D-Volumenrekonstruktion Histologie. Während 2D-Histologie Bilder zeigen die strukturellen Unterschiede zwischen Paaren von Abschnitten, bietet 3D-Histologie Volumen die Möglichkeit, die Unterschiede in der Form und Volumen der Brustdrüsen zu visualisieren.

Ein Fallstrick der traditionellen Mikroskopie-Techniken ist, dass das Verständnis von einem Organ auf der mikroskopischen Ebene wird auf einem Feld-of-view in einer Zeit beschränkt. Auch &quot;total Offenlegung&quot; Dias, die gesamte Folie Abschnitte bieten, scheitern, um dreidimensionale Informationen. Mit der Entwicklung des gesamten Objektträger, dynamische Scan-Technologien, unsere Fähigkeit, einen Abschnitt in seiner Gesamtheit zu sehen gestiegen ist, aber die Extrapolation Strukturen erfordert 3D-Histologie V...

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.

Die Rekonstruktion der 3-Dimensional Histologie Volume und seine Anwendung auf Maus Milchdrüsen Studieren

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 Aufrufe.  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. 

Serie: 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.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

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

Forschung

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.

Die Leitersprosse Walking-Aufgabe: Ein Scoring-System und seine praktische Anwendung.

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.

Objektiv Transplantation in Zebrafisch und ihre Anwendung in der Analyse von Eye-Mutanten

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.

Maus Brustepithelzellen Form Mammospheres Während lactogenic Differenzierung

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 der Brustdrüse Entwicklung und Funktion in Mausmodellen

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.

Intraduktale Injection für Lokalisierte Drug Delivery auf die Maus Milchdrüse

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.

Ein Fluoreszenz-basierte Exonuklease Assay zu DmWRNexo, Ortholog der Menschen Progeroid WRN Exonuclease, und ihre Anwendung auf andere Nukleasen Charakterisieren

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

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

Anwendung von Retinsäure zu Osteozyten Kulturen aus primären Maus-Osteoblasten erhalten

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

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.

Hochdurchsatz-Analyse von Bild Tumorsphäroide: Eine benutzerfreundliche Software-Anwendung, um die Größe der Sphäroide automatisch und präzise messen

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

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.

Mess Druck Volumen Loops in der Maus

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