Ich danke Dr. Alan O. Perantoni (CDBL/NCI/NIH) für die Einreichung des Manuskripts zu unterstützen. Ich danke auch Dr. Michael Hall (CDBL/NCI/NIH) und Nirmala Sharma (CDBL/NCI/NIH) für die Bearbeitung dieser Handschrift. Ich bin dankbar für seine ausgezeichnete Maus Tierhaltung, Lai Thang (SAIC). Diese Arbeit wurde von den National Institutes of Health, National Cancer Institute und Center for Cancer Research unterstützt.

Mäuse (Wnt5a Flox / Flox Mäuse (Wnt5a tm1.1Tpy) und Dll1Cre Linie, UVJO-Maus-Modell) 7 wurden nach NIH-Richtlinien für die Pflege und Verwendung von Labortieren verwaltet und studierte unter ein Protokoll durch die NCI-Frederick Animal Care and Use Committee genehmigt.
 1. Vorbereitung des Methylenblau Farbstofflösung <ol><li>0,1 g Methylenblau Pulver zu messen.</li>
	<li>Lösen sich die Methylenblau in 10 mL normale Kochsalzlösung oder 1 x Phosphat gepufferte Kochsalzlösung (PBS) vollständig durch vortexen.</li>
	<li>Filtern die 10 mg/mL Methylenblau-Lösung mit einer Spritze Filter (Membran Porengröße 0.45µm) zu beseitigen, Verstopfungen beim Einspritzen.</li>
	<li>Montieren einer sterilen Kopfhaut Vene Set (27GX3/4 &#8243;) mit 3 mL Einwegspritze und füllen Sie die Spritze mit 3 mL Methylenblau-Lösung gefiltert. 
	Hinweis: Zur Vermeidung von hydrostatischen Druck und nachfolgenden Farbstoff Fluss legen Sie die Spitze der Nadel über die Spritze gefüllt mit Methylenblau-Lösung.</li>
</ol> 2. Dissektion der pränatalen Embryonen <ol><li>sauber sezieren, Scheren und Pinzetten mit 70 % igem Ethanol.</li>
	<li>, Perinatale Embryonen im E18.5 oder E19.5, sammeln einschläfern schwangere Mäusen zuerst mit CO 2 Einatmen und zervikale Dislokation nach NIH-Richtlinien führen. 
	Hinweis: Die schwangere weibliche Maus in der Regel gebiert E18.5 ab, jedoch größere Nieren sind leichter zu manipulieren. Daher sind die Nieren im E19.5 näher zur Geburt leichter auf diese Analyse durchzuführen. Aber Welpen mit bilateralen Harnwege Hindernisse sterben nach der Geburt. Abhängig von den Eigenschaften der experimentellen Mäuse, sollte eine entsprechende Sammlung Wochentag/Uhrzeit empirisch ermittelt werden.</li>
	<li>70 % igem Ethanol auf der ventralen Bauch-Oberfläche sprühen und öffnen Sie dann die Bauchhöhle mit ventral sezierenden Schere und Zange.</li>
	<li>Heben die gesamte Gebärmutter und trennen Sie ihn aus dem Körper durch Schneiden mit Präparierscheren an den Spitzen der uterinen Hörner.</li>
	<li>Spülen die gesamte Gebärmutter mit 1 X PBS in einer Petrischale.</li>
	<li>Die Gebärmutter segmentartig mit Präparierscheren schneiden und entfernen Sie plazentar Decidua mit sezieren Zange um die Embryonen im Dotter Sacs freizulegen.</li>
	<li>Der Dottersack erst dann entfernen und die amniotische Membrane mit sezieren Zange um die perinatalen Embryonen zu befreien.</li>
	<li>Enthaupten einen Embryo mit Präparierscheren. Wischen Sie überschüssiges Blut mit steriler Gaze-Pads. Festnageln des Embryos mit der ventralen Oberfläche bis auf einen sezierenden Mikroskop, ausgestattet mit einer digitalen Kamera für die Bildgebung. Seine Rute für die Genotypisierung zu sammeln, wenn nötig. 
	Hinweis: Führen Sie Injektionen ein Embryo zu einem Zeitpunkt.</li>
	<li>Öffnen Sie vorsichtig die Bauchhöhle des Embryos mit der Pinzette durch Aufreißen der Haut. Dann sorgfältig entfernen Sie überschüssige Organe und Gewebe wie Leber, Magen und Darm mit der Pinzette durch Schneiden oder herausziehen um zu entlarven, die Nieren, die Harnleiter und die Blase, die dorsal befinden ( Abbildung 1A). Absorbieren überschüssiges Blut aus den seziert Embryo mit steriler Gaze-Pads, wenn nötig. 
	Hinweis: Überschüssige Blut stört bei der Identifizierung von Nierenbecken für die Injektion von Farbstoff.</li>
</ol> 3. Injektion von Methylenblau-Dye in das Nierenbecken und Überwachung fließen Dye <ol><li>Entfernen Luftblasen in die Nadel und Schlauch durch Ausweisung Methylenblau-Lösung aus der Nadelspitze durch hydrostatischen Druck. Heben Sie die Spritze mit der Farbstofflösung über dem Niveau der Nadelspitze zu fließen beginnen, und senken Sie dann die Spritze zu unterbrechen.</li>
	<li>Stechen Sie die Nadel in das Nierenbecken in der Nähe von den proximalen Harnleiter, kümmert sich nicht um ihn einmal stören. Dye Injektion in eine Niere zu bestimmen, Obstruktion der Harnwege führen. 
	Hinweis: Führen Sie färben Injektion in jede abnorme Nieren 7 ersten folgen die gleiche Weise zu bestimmen, seine Harnwege Obstruktion.</li>
	<li>Heben die Spritze bis etwa 20 cm hydrostatischen Druck und 15 µL - 60 µL Farbstoff Lösung fließen zu lassen. Die Durchflussmenge wird über 3 µL/s sein, wenn die Spritze zu dieser Höhe oberhalb des Embryos ausgelöst wird.</li>
	<li>Überwachen die blaue Farbe des Farbstoffs zuerst in das Nierenbecken und dann in der Länge von den Harnleiter und schließlich in die Blase Lumen. 
	Hinweis: Es dauert ca. 5 s zu sehen, eine schwache Farbe innerhalb der Blase Lumen entstehen, wenn die Harnleiter in die Blase richtig eingelegt ist. Den Farbstoff der Website blockiert ca. 15 ansammeln lassen s erscheint keine Farbe in die Blase Lumen.</li>
	<li>Legen Sie die Spritze bis Haltestelle Farbstoff Strömung und ziehen Sie die Nadel aus der Niere.</li>
	<li>Nehmen Sie Bilder von der Niere, der Harnleiter und der Blase mit Farbstofflösung verfolgt mit der Kamera und imaging-Programm verbunden zu einem Stereomikroskop.</li>
	<li>Notieren Hydronephrose der Niere, Hydroureter und die endgültige Position der Farbstofflösung in einem Labor Notebook.</li>
	<li>Perform Injektion der kontralateralen Niere wie beschrieben in Abschnitt 3.1), 3.5) und ermöglichen Farbstoff Fluss etwa 20 s insgesamt. 
	Hinweis: Die Blase Lumen wird mit Farbstofflösung, durch eine starke blaue Farbe angezeigt, wenn der Harnleiter in die Blase Lumen richtig eingelegt ist gefüllt werden. Nach Bewertung der Harnwege Kreuzung Obstruktion, die Harnleiter und die Blase für Schnitt fixiert werden können.</li>
</ol>

<ol>
	<li>Rasouly, H. M., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+urinary+tract+development+and+disease.">Lower urinary tract development and disease.</a> Wiley Interdiscip Rev Syst Biol Med. 5, 307-342 (2013).</li><li>Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Box+Genes+in+the+Kidney+and+Urinary+Tract.">T-Box Genes in the Kidney and Urinary Tract.</a> Curr Top Dev Biol. 122, 245-278 (2017).</li><li>Bohnenpoll, T., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureter+growth+and+differentiation.">Ureter growth and differentiation.</a> Semin Cell Dev Biol. 36, 21-30 (2014).</li><li>Nicolaou, N., Renkema, K. Y., Bongers, E. M., Giles, R. H., Knoers, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic,+environmental,+and+epigenetic+factors+involved+in+CAKUT.">Genetic, environmental, and epigenetic factors involved in CAKUT.</a> Nat Rev Nephrol. 11, 720-731 (2015).</li><li>Tripathi, P., Wang, Y., Casey, A. M., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+canonical+Smad+signaling+in+ureteral+and+bladder+mesenchyme+causes+ureteropelvic+junction+obstruction.">Absence of canonical Smad signaling in ureteral and bladder mesenchyme causes ureteropelvic junction obstruction.</a> J Am Soc Nephrol. 23, 618-628 (2012).</li><li>Aoki, Y., 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=Id2+haploinsufficiency+in+mice+leads+to+congenital+hydronephrosis+resembling+that+in+humans.">Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans.</a> Genes Cells. 9, 1287-1296 (2004).</li><li>Yun, K., Perantoni, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydronephrosis+in+the+Wnt5a-ablated+kidney+is+caused+by+an+abnormal+ureter-bladder+connection.">Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection.</a> Differentiation. 94, 1-7 (2017).</li><li>Uetani, N., Bouchard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plumbing+in+the+embryo:+developmental+defects+of+the+urinary+tracts.">Plumbing in the embryo: developmental defects of the urinary tracts.</a> Clin Genet. 75, 307-317 (2009).</li><li>Murawski, I. J., Watt, C. L., Gupta, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+urinary+tract+defects+in+mice:+methods+to+detect+the+presence+of+vesicoureteric+reflux+and+urinary+tract+obstruction.">Assessing urinary tract defects in mice: methods to detect the presence of vesicoureteric reflux and urinary tract obstruction.</a> Methods Mol Biol. 886, 351-362 (2012).</li><li>Weiss, A. C., 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=Nephric+duct+insertion+requires+EphA4/EphA7+signaling+from+the+pericloacal+mesenchyme.">Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.</a> Development. 141, 3420-3430 (2014).</li></ol>

Der Autor hat nichts preisgeben.

Maus-Nieren sind funktionale Beginn E16.5 und ein Farbstoff-Injektion-Test ist ab diesem Zeitpunkt theoretisch möglich. Jedoch die Niere ist zu klein, um mit der Farbstofflösung injiziert werden und Phänotypen wie Hydronephrose und Hydroureter sind nicht eindeutig beobachtet, da diese Phänotypen als Nebeneffekt des Urins durch abnorme Urin Transport aufgebaut sind. Diese Phänotypen von Hindernissen wie UPJO oder UVJO, sind offensichtlich am E18.5 durch aufgeblähten Nieren und Harnleiter. Größere Nieren von Embryo...

Das Harnsystem besteht aus einem Paar Nieren und Harnleiter und einer gemeinsamen Blase und Harnröhre. Die Hauptfunktion des Harnsystems ist Körper-Homöostase durch die Verwaltung der Wasser- und Elektrolyt-Gleichgewicht des Blutes. Die Nieren filtern das Blut zur Kontrolle Elektrolyt Konzentrationen und Säure-Basen-Gleichgewicht und produzieren Urin um überschüssiges Wasser und Abfällen einschließlich Solute und Stoffwechselprodukte auszuscheiden. Urin wird dann durch den Harnleiter aus dem Nierenbecken der Niere, die Bladderin einer unidirektionalen Weise transportiert, wo es gespeichert und schließlich über die Harnröhre1beseitigt.
Die Harnleiter sind gerade Rohre mit Ursprung aus dem nephric Rohr. Nach angehenden aus dem nephric Rohr an embryonalen Tag 10.5 (E10.5) in der Maus, der Harnleiter Stiel verlängert und differenziert in eine mehrschichtige Struktur namens Urothel, die zwischen Maus undurchlässig ist, E12.5 und E16.5. Die mesenchymalen Zellen umgibt den Harnleiter Stiel unterscheiden sich auch in drei Schichten bestehend aus inneren Stromazellen Zellen, zwischen dicken glatten Muskelzellen und äußeren adventitial Fibroblasten. Harnleiter peristaltische Wellen, Einleitung in das Nierenbecken werden vermehrt durch die glatte Muskulatur Schicht der Mauer Harnleiter in die Blase Urin2,3, transportieren die ab E16.5 in der Maus-1produziert wird.
Angeborene Fehlbildungen der Nieren und Harnwege (CAKUT) gehören zu den häufigsten Erbkrankheiten, bei rund 1 % der menschlichen Föten1,4, und setzt sich aus einer Vielzahl von Phänotypen einschließlich Hydronephrose und Hydroureter. Die abnorme Ansammlung von Urin in der Niere und Harnleiter führt Hydronephrotic Nieren- und Hydroureter Bildung. Eine Ursache der Hydronephrose und/oder Hydroureter Formation ist eine Obstruktion der Harnwege. Ureteropelvic Kreuzung Obstruktion (UPJO), verursacht durch aberrante Harnfluss durch eine Blockade zwischen den proximalen Harnleiter und das Nierenbecken, wodurch Hydronephrose und proximale Hydroureter Verengung mit Winkelung oder hartnäckige Falten5, 6. Darüber hinaus wird die ektopische Einfügung von den distalen Harnleiter in die Blasenwand oder die Fortpflanzungsorgane vesikale Kreuzung Obstruktion (UVJO) genannt. UVJO kann auch Hydronephrose und Hydromegaureter Bildung7,8induzieren. Ein zusätzliche vesikale Kreuzung (UVJ) defekt ist Vesicoureteric Reflux (VUR). VUR zeichnet sich durch retrograde Urin-Flow aus der Blase in Richtung Niere bei UVJ. Im Vergleich zu UVJO, zeigen perinatale Embryonen mit VUR deutlich einen aufgeblähten Hydronephrotic Nieren- oder schweren Hydroureter Phänotyp9nicht.
In der Labormaus kann Urin-Flow durch Injektion eines Farbstoffes, wie Methylenblau, in das Nierenbecken9untersucht werden. Die Injectedmethylene blaue Lösung wird die Flugbahn der Urin aus dem Nierenbecken durch den Harnleiter und in die Blase verfolgen. Hydronephrose kann durch eine Erweiterung des Farbstoffs in der Niere erkannt werden. UPJO kann als eine Blockade der Farbstoff Strömung an den proximalen Harnleiter mit aufgeblähten Nierenbecken5erkannt werden. Dilatation der Harnleiter durch einen aufgeweiteten Durchmesser angegeben zeigt ein Beispiel für Hydroureter. Zu guter Letzt Farbstoff Ansammlung an der Blasenwand oder auf der Website der Fortpflanzungsorgane zeigt UVJO mit aufgeblähten Hydronephrotic Niere und geweitet Hydromegaureter7,10. Um VUR zu erkennen, die Farbstofflösung in die Blase injiziert und anschließende retrograde wird in der Niere9überwacht.
Hier ist ein Protokoll für Methylenblau färben Injektion in das Nierenbecken eines perinatalen Embryos präsentiert. Dieses Protokoll ermöglicht die Rückverfolgung von Urin-Flow aus dem Nierenbecken durch den Harnleiter und in die Blase und prüft mögliche Harnwege Kreuzung Hindernisse wie UPJO oder UVJO.

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assessing urinary tract junction obstruction defects by methylene blue dye injection

Harnwege Kreuzung Obstruktion Mängel sind angeborene Anomalien induzierende Hydronephrose und Hydroureter. Murine Harnwege Kreuzung Obstruktion Mängel können beurteilt werden, durch die Verfolgung Methylenblau färben Strömung innerhalb der Harnwege. Methylenblau-Dye in das Nierenbecken der perinatalen embryonale Nieren injiziert und Farbstoff wird überwacht von den Nierenbecken der Niere durch den Harnleiter und in das Lumen der Blase nach dem hydrostatischen Druck anwenden. Farbstoff Anhäufung werden deutlich in der Blase Lumen der normalen perinatale Harnwege, sondern wird zwischen dem Nierenbecken und den Endpunkt der eine abnorme Harnleiter, eingeschränkt werden, wenn die Harnwege Hindernisse auftreten. Diese Methode ermöglicht die Bestätigung der Harnwege Kreuzung Hindernisse und Visualisierung von Hydronephrose und Hydroureter. Dieses Manuskript beschreibt ein Protokoll für Methylenblau färben Injektion in das Nierenbecken, Harnwege Kreuzung Hindernisse zu bestätigen.

Die Nieren und unteren Harnwege befinden sich dorsal zu den meisten anderen inneren Organe wie Leber und Darm. Nach dem Entfernen dieser andere innere Organe, sind ein paar Nieren und Harnleiter und einer einzelnen Blase sichtbar (Abb. 1A). Auf erfolgreiche Dissektion fließt der Farbstoff injiziert das Nierenbecken in die Blase über den Harnleiter von der Niere. Das Nierenbecken ist der trichterartige dilatative proximalen Teil der Harnleiter in die Niere. ...

Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Methylenblau-Dye-Injektion in das Nierenbecken erleichtert die Beurteilung der Harnwege Kreuzung Obstruktion Mängel während der Maus embryonalen Harnwege Entwicklung. Hier wird ein Protokoll für Methylenblau-Dye-Injektion in das Nierenbecken beschrieben.

Beurteilung der Harnwege Junction Obstruktion Mängel durch Methylenblau-Dye-Injektion

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...

assessing-urinary-tract-junction-obstruction-defects-methylene-blue

Research

JoVE Journal

Biology

15.6K Aufrufe.  National Institutes of Health. Methylenblau-Dye-Injektion in das Nierenbecken erleichtert die Beurteilung der Harnwege Kreuzung Obstruktion Mängel während der Maus embryonalen Harnwege Entwicklung. Hier wird ein Protokoll für Methylenblau-Dye-Injektion in das Nierenbecken beschrieben.

Serie: Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissecting und Aufnahme von C. elegans neuromuskulären Synapse

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Forschung

Biologie

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafisch Gehirn Ventrikel Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Die Implantation von Ferumoxides Labeled humanen mesenchymalen Stammzellen in Knorpeldefekte

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi-Interferenz durch dsRNA Injektion in Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Ein Maus-Modell der teilweisen Darmverschluss

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Ein Jodid-gelb fluoreszierende Protein-Gap Junction-interzelluläre Kommunikation Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Ex Vivo-Methode zur Beurteilung der spontanen Motilität des Mausreproduktivtrakts und eines MATLAB-basierten Uterus Motion Tracking Algorithmus für die Datenanalyse

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...