Ringrazio il dottor Alan O. Perantoni (NIH/NCI/CDBL) per sostenere la presentazione di questo manoscritto. Ringrazio anche il Dr. Michael Hall (NIH/NCI/CDBL) e Nirmala Sharma (NIH/NCI/CDBL) per la modifica di questo manoscritto. Sono grato a Lai Thang (SAIC) per il suo allevamento eccellente mouse. Questo lavoro è stato sostenuto dagli istituti nazionali di salute, National Cancer Institute e centro per la ricerca sul cancro.

topi (Wnt5a flox / flox topi (Wnt5a tm1.1Tpy) e Dll1Cre linea, modello murino UVJO) 7 erano gestiti secondo le linee guida NIH per la cura e l'uso degli animali da laboratorio Studiò con un protocollo approvato dal comitato di uso e NCI-Frederick Animal Care.
 1. preparazione della soluzione di colorante blu di metilene <ol><li>misura 0,1 g di blu di metilene polvere.</li>
	<li>Sciogliere completamente il blu di metilene in salino normale 10 mL o 1 x tamponato fosfato salino (PBS) nel Vortex.</li>
	<li>Filtrare la soluzione di blu di metilene 10 mg/mL con un siringa filtro (dimensione dei pori di membrana 0.45µm) per eliminare l'intasamento durante l'iniezione.</li>
	<li>Montare un sterile del cuoio capelluto vena set (27GX3/4 &#8243;) con una siringa monouso da 3 mL e riempire la siringa con 3 mL di filtrato soluzione di blu di metilene. 
	Nota: Per evitare successive tintura flusso e pressione idrostatica, posizionare la punta dell'ago sopra la siringa riempita con soluzione di blu di metilene.</li>
</ol> 2. Dissezione di embrioni prenatale <ol><li>pulito di dissezione Forbici e pinze con etanolo al 70%.</li>
	<li>Per raccogliere embrioni perinatali a E18.5 o e 19.5, eutanasia topi incinti prima mediante inalazione di CO 2 e quindi eseguire dislocazione cervicale secondo linee guida NIH. 
	Nota: Il mouse femmina incinto solitamente dà vita a partire da E18.5, tuttavia, i reni più grandi sono più facili da manipolare. Di conseguenza, reni a e 19.5 più vicino alla nascita sono più facili da eseguire questa analisi su. Tuttavia, cuccioli con bilaterale delle vie urinarie ostruzioni muoiono dopo la nascita. A seconda delle caratteristiche di topi sperimentali, una raccolta appropriata giorno e ora dovrebbe essere determinata empiricamente.</li>
	<li>Spray etanolo al 70% sulla superficie addominale ventrale e quindi aprire la cavità addominale ventrale utilizzando pinze e forbici per dissezione.</li>
	<li>Sollevare l'intero utero e separarla dal corpo di taglio con forbici alle punte dei corni uterini di dissezione.</li>
	<li>Sciacquare l'intero utero con PBS 1X in una capsula Petri.</li>
	<li>Tagliare l'utero segmentally con forbici di dissezione e rimuovere placenta decidua con forcipe per esporre gli embrioni nei sac di tuorlo di dissezione.</li>
	<li>Rimuovere il sacco vitellino prima e poi rimuovere la membrana amniotic con forcipe per liberare gli embrioni perinatali di dissezione.</li>
	<li>Decapitare un embrione con forbici di dissezione. Pulire il sangue in eccesso con compresse di garza sterile. L'embrione pin con la sua superficie ventrale up in un microscopio per dissezione equipaggiato con una fotocamera digitale per l'imaging. Raccogliere la sua coda per genotipizzazione se necessario. 
	Nota: Eseguire un embrione iniezioni alla volta.</li>
	<li>Aprire con cautela la cavità addominale di un embrione con forcipe strappando la pelle. Quindi, rimuovere con delicatezza in eccesso organi e tessuti quali fegato, stomaco e intestino con forcipe tagliandoli o tirarle fuori per esporre i reni, ureteri e vescica che sono situati dorsalmente ( Figura 1A). Assorbire il sangue in eccesso dall'embrione dissecata con compresse di garza sterile se necessario. 
	Nota: Il sangue in eccesso interferisce con identificazione della pelvi renale per l'iniezione di colorante.</li>
</ol> 3. Iniezione di colorante blu di metilene nella pelvi renale e monitoraggio del flusso di tintura <ol><li>Rimuovi bolle nell'ago e la tubazione espellendo soluzione di blu di metilene dalla punta dell'ago dalla pressione idrostatica. Sollevare la siringa contenente la soluzione di colorante sopra il livello della punta dell'ago per avviare il flusso e quindi abbassare la siringa per interrompere il flusso.</li>
	<li>Inserire l'ago nella pelvi renale vicino l'uretere prossimale, facendo attenzione a non per disturbare una volta posizionati. Eseguire l'iniezione della tintura in un rene per determinare la sua ostruzione dell'apparato urinario. 
	Nota: Eseguire la tintura prima iniezione in qualsiasi renale anormale 7 seguendo allo stesso modo per determinare la sua ostruzione dell'apparato urinario.</li>
	<li>Sollevare la siringa fino a circa 20 cm per fornire pressione idrostatica e lasciare 15 µ l - 60 µ l del flusso di soluzione del colorante. La portata sarà circa 3 µ l/s se la siringa è aumentata a questa altezza sopra l'embrione.</li>
	<li>Monitorare il colore blu della tintura a partire prima nella pelvi renale, poi nella lunghezza dell'uretere e infine nel lume della vescica. 
	Nota: Si impiegano circa 5 s per vedere un colore tintura debole emergono all'interno del lumen della vescica se l'uretere è correttamente inserito nella vescica. Consentire il colorante ad accumularsi nel sito bloccato circa 15 s se nessun colore tintura compare nel lume della vescica.</li>
	<li>Posizionare la siringa fino a fermata tintura flusso e rimuovere l'ago dal rene.</li>
	<li>Foto per scattare foto di rene, uretere e vescica tracciata con soluzione colorante con la fotocamera e programma di imaging collegato a uno stereomicroscopio.</li>
	<li>Fare un record di idronefrosi del rene, hydroureter e la posizione finale della soluzione colorante in un quaderno di laboratorio.</li>
	<li>Eseguire l'iniezione di rene controlaterale come descritto nelle sezioni 3.1) a 3.5) e consentire il flusso di tintura circa 20 s in totale. 
	Nota: Il lume della vescica sarà riempito di soluzione colorante, indicata da un colore blu forte, se l'uretere è inserito correttamente nel lume della vescica. Dopo la valutazione dell'ostruzione della giunzione delle vie urinarie, ureteri e vescica può essere fissate per sezionamento.</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>

L'autore non ha nulla di divulgare.

I reni mouse sono inizio funzionale alle E16.5 e una prova di iniezione della tintura è teoricamente possibile da questo punto di tempo. Tuttavia, il rene è troppo piccolo per essere iniettato con la soluzione colorante e fenotipi quali idronefrosi e hydroureter non si osservano chiaramente dal momento che questi fenotipi sono un effetto secondario di urina accumulato a causa del trasporto delle urine anormali. Questi fenotipi, da ostruzioni quali UPJO o UVJO, sono evidenti alle E18.5 di dilatato reni e ureteri. Reni p...

Il sistema urinario è costituito da una coppia di reni e ureteri e un comune della vescica e dell'uretra. La funzione principale del sistema urinario è di mantenere l'omeostasi corporea gestendo l'equilibrio di acqua ed elettroliti del sangue. I reni filtrano il sangue per controllo di concentrazione di elettroliti e l'equilibrio acido-base e producono urina per espellere l'acqua in eccesso e rifiuti tra cui soluti e metaboliti. Urina viene quindi trasportato attraverso l'uretere dalla pelvi renale del rene per la bladderin un modo unidirezionale dove viene immagazzinata e infine eliminata attraverso l' uretra1.
Gli ureteri sono tubi dritti che proviene dal dotto nephric. Dopo in erba dal dotto nephric al giorno embrionale 10.5 (e 10.5) nel topo, il gambo di uretere si allunga e differenzia in una struttura multistrata denominata urothelium che è impermeabile tra mouse 12.5 ed E16.5. Le cellule mesenchimali che circondano il gambo dell'uretere sono anche differenziate in tre strati composto da cellule stromali interne, cellule di muscolo liscio spessore intermedio e fibroblasti adventitial esterni. Le onde peristaltiche ureterale avvio nella pelvi renale vengono propagate attraverso lo strato di muscolo liscio della parete dell'uretere alla vescica per il trasporto di urine2,3, che è prodotto a partire da E16.5 in mouse1.
Anomalie congenite del rene e delle vie urinarie (CAKUT) sono tra le più frequenti malattie genetiche, presente in circa 1% dei feti umani1,4e composto di una varietà di fenotipi compreso idronefrosi e hydroureter. L'abnorme accumulo di urina nel rene e uretere provoca hydronephrotic del rene e hydroureter formazione. Una causa di formazione di idronefrosi e/o hydroureter è un'ostruzione delle vie urinarie. Ureteropelvic della giunzione (UPJO) è causato dal flusso di urina aberrante a causa di un blocco tra l'uretere prossimale e la pelvi renale, con conseguente idronefrosi e prossimale hydroureter restringimento con angolazione o persistente pieghevole5, 6. Inoltre, l'inserimento ectopica dell'uretere distale nella parete della vescica o del tratto riproduttivo viene chiamato ostruzione giunzione ureterovesical (UVJO). UVJO può anche indurre l'idronefrosi e hydromegaureter formazione7,8. Un difetto di giunzione (UVJ) ulteriori ureterovesical è reflusso vesicoureteric (RVU). VUR è caratterizzata da flusso retrogrado dell'urina dalla vescica verso il rene presso UVJ. Rispetto al UVJO, perinatali embrioni con VUR non distintamente mostrano un rene hydronephrotic dilatato o grave hydroureter fenotipo9.
Nel topo di laboratorio, il flusso di urina può essere esaminato tramite l'iniezione di un colorante, come il blu di metilene, nel bacino renale9. La soluzione di injectedmethylene blu si traccia la traiettoria di urina dalla pelvi renale attraverso l'uretere e la vescica. Idronefrosi possono essere riconosciuto da un'espansione del colorante nel rene. UPJO può essere rilevato come un blocco del flusso di tintura all'uretere prossimale con pelvi renale dilatata5. Qualsiasi dilatazione dell'uretere indicato da un diametro allargato di seguito viene illustrato un esempio di hydroureter. Infine, accumulo di colorante alla parete della vescica o presso il sito del tratto riproduttivo indica UVJO con rene hydronephrotic dilatato e dilatato hydromegaureter7,10. Per rilevare VUR, la soluzione colorante viene iniettata nella vescica e conseguente flusso retrogrado è monitorato nel rene9.
Qui, un protocollo per l'iniezione di colorante blu di metilene nella pelvi renale di un embrione perinatale è presentato. Questo protocollo permette l'analisi del flusso di urina dalla pelvi renale attraverso l'uretere e la vescica e verifica potenziali ostacoli di giunzione delle vie urinarie come UPJO o 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

Difetti di ostruzione delle vie urinarie giunzione sono anomalie congenite inducendo idronefrosi e hydroureter. Difetti di ostruzione giunzione murino delle vie urinarie possono essere valutati da monitorando il flusso di colorante blu di metilene all'interno del sistema urinario. Blu di metilene colorante viene iniettato nella pelvi renale dei reni embrionali perinatale e flusso di tintura è monitorato dalla pelvi renale del rene attraverso l'uretere e nel lume della vescica dopo l'applicazione di pressione idrostatica. Accumulo di colorante sarà evidente nel lume della vescica dell'apparato urinario perinatale normale, ma sarà vincolata tra pelvi renale e il punto finale di un uretere anormale, in caso di ostruzioni delle vie urinarie. Questo metodo facilita la conferma delle ostruzioni di giunzione delle vie urinarie e visualizzazione di idronefrosi e hydroureter. Questo manoscritto descrive un protocollo per l'iniezione della tintura del blu di metilene nella pelvi renale per confermare ostruzioni di giunzione delle vie urinarie.

I reni e il sistema urinario inferiore si trovano dorsale alla maggior parte degli altri organi interni come il fegato ed intestino. Dopo la rimozione di questi altri organi interni, una coppia di reni e ureteri e vescica una singola sono visibili (Figura 1A). Al momento di successo dissezione, il colorante iniettato nella pelvi renale fluirà dal rene nella vescica tramite l'uretere. La pelvi renale è la parte prossimale dilatata imbuto dell'uretere nel ren...

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

Iniezione della tintura del blu di metilene nella pelvi renale facilita la valutazione dei difetti di ostruzione delle vie urinarie giunzione durante lo sviluppo embrionale dell'apparato urinario del mouse. Qui, un protocollo per l'iniezione della tintura del blu di metilene nella pelvi renale è descritto.

Valutazione difetti di ostruzione delle vie urinarie Junction iniettando colorante blu di metilene

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 Views.  National Institutes of Health. Iniezione della tintura del blu di metilene nella pelvi renale facilita la valutazione dei difetti di ostruzione delle vie urinarie giunzione durante lo sviluppo embrionale dell'apparato urinario del mouse. Qui, un protocollo per l'iniezione della tintura del blu di metilene nella pelvi renale è descritto.

Video: 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.

Topo iniezione parete della vescica

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

Ricerca

Medicina

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).

L'applicazione dell'1% di colorazione blu di metilene come singola tecnica nel cancro al seno 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 ...

Ricerca sul cancro

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.

Cistografia fotoacustico

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

Bioingegneria

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.

Rilevazione intraoperatoria di endometriosi sottile: un nuovo paradigma per la rilevazione e il trattamento del dolore pelvico associato con la perdita di integrità peritoneale

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-based Endomicroscopy laser confocale del tratto urinario: La Tecnica

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.

Procedura transuretrale instillazione in di topo maschio adulto

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

Immunologia e infezioni

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).

Ultrasonografia del tratto urinario maschile adulto per test funzionali urinari

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.

Utilizzo della microscopia a 2 fotoni per quantificare gli effetti dell'ostruzione ureterale unilaterale cronica sui processi glomerulari

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

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.
In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

In vivo Misurazione luminale del rilascio uroteliale di ATP evocato dalla distensione nei roditori

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

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.

Un modello murino immaturo di ostruzione ureterale unilaterale reversibile

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