Name: assessing urinary tract junction obstruction defects by methylene blue dye injection
Uploaded: 2017-10-12T12:30:00.000+00:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

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.

<table><tbody><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140</td><td></td></tr><tr><td>Quality Biological Inc. NORMAL SALINE - 500ML</td><td>Fisherscientific</td><td>50-983-204</td><td></td></tr><tr><td>3 mL disposable syringe with BD Luer-Lok tip</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Acrodisc Syringe Filters with Supor Membrane</td><td>Pall corporation</td><td>4614</td><td></td></tr><tr><td>Exel Scalp Vein (Butterfly) Sets; 27G x 3/4&quot; 12&quot;&amp;nbsp;</td><td>EXELINT</td><td>26709</td><td></td></tr><tr><td>Delicate Operating Scissors</td><td>Roboz Surgical Instrument Co.</td><td>RS-6702</td><td></td></tr><tr><td>Micro Dissecting Forceps</td><td>Roboz Surgical Instrument Co.</td><td>RS-5135</td><td></td></tr><tr><td>Dumont Tweezers; Pattern #5</td><td>Roboz Surgical Instrument Co.</td><td>RS-5045</td><td></td></tr><tr><td>BD PrecisionGlide Needles 30 G x 1/2&quot;</td><td>BD</td><td>305106</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

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

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Maus Blasenwand Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Forschung

Medizin

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

Die Anwendung von 1% Methylen blauer Farbstoff als eine einzige Technik in Brustkrebs Sentinel Node Biopsie

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

Krebsforschung

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Bewertung von Biomaterialien für Blasenvergrößerung mit Zystometrische Analysen in verschiedenen Tiermodellen

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Biotechnik

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Photoakustische Zystographie

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Intraoperative Detektion von Subtle Endometriosis: A Novel Paradigm for Detection und Behandlung von Pelvic Pain mit dem Loss of Peritoneal Integrity-Zustellbare

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Probe-basierte konfokale Laser Endomikroskopie der Harnwege: Die Technik

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

Transurethrale Instillation Verfahren bei erwachsenen männlichen Maus

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

Immunologie und Infektion

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

Ultrasonographie des adulten männlichen Harntraktes für Harnfunktionstests

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Mit der 2-Photonen-Mikroskopie die Auswirkungen einer chronischen einseitigen Harnleiterobstruktion auf glomeruläre Prozesse quantifizieren

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

Ein unreifes Mausmodell einer reversiblen einseitigen Harnleiterobstruktion

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...