Agradeço o Dr. Alan O. Perantoni (CDBL/ICN/NIH) para apoiar a apresentação deste manuscrito. Agradeço também a Dr. Michael Hall (CDBL/ICN/NIH) e Nirmala Sharma (CDBL/ICN/NIH) para editar este manuscrito. Eu sou grato a Lai Thang (SAIC) para sua criação do mouse excelente. Este trabalho foi financiado pelos institutos nacionais de saúde, Instituto Nacional de câncer e o centro para pesquisa do câncer.

ratos (Wnt5a flox / ratos flox (Wnt5a tm1.1Tpy) e Dll1Cre linha, modelo de mouse UVJO) 7 eram geridas de acordo com as diretrizes do NIH para o cuidado e o uso de animais de laboratório e estudado sob um protocolo aprovado pelo Comitê de uso e cuidado do Animal de NCI-Frederick.
 1. preparação da solução de corante azul de metileno <ol><li>medir pó de azul de metileno 0,1 g.</li>
	<li>Dissolver o azul de metileno em solução salina normal de 10 mL ou 1 x salina tamponada fosfato (PBS) completamente vortexing.</li>
	<li>A 10 mg/mL solução de azul de metileno de filtragem com um filtro de seringa (tamanho dos poros da membrana 0.45µm) para eliminar a obstrução durante a injeção.</li>
	<li>Montar um escalpe estéril veia conjunto (27GX3/4 &#8243;) com uma seringa descartável de 3 mL e encha a seringa com 3ml filtrado solução de azul de metileno. 
	Nota: Para evitar a pressão hidrostática e fluxo subsequente tintura, coloque a ponta da agulha acima a seringa preenchida com solução de azul de metileno.</li>
</ol> 2. Dissecação de pré-natal de embriões <ol><li>limpo, dissecando a tesoura e pinça com etanol a 70%.</li>
	<li>Para coletar embriões perinatais em E18.5 ou E19.5, eutanásia de ratos grávidas primeiro usando a inalação de CO 2 e então executar o deslocamento cervical de acordo com as diretrizes do NIH. 
	Nota: O rato fêmea grávido geralmente dá à luz partida no E18.5, no entanto, os rins maiores são mais fáceis de manipular. Portanto, os rins em E19.5 perto de nascimento são mais fáceis de realizar esta análise em. No entanto, filhotes com bilateral do trato urinário obstruções morre após o nascimento. Dependendo das características dos ratos experimentais, uma dia e hora de recolha deve ser determinada empiricamente.</li>
	<li>Spray etanol a 70% na superfície ventral abdominal e então abrir a cavidade abdominal ventralmente usando dissecação tesoura e pinça.</li>
	<li>Levantar o útero inteiro e separá-lo do corpo cortando com tesoura nas pontas dos cornos uterinos de dissecação.</li>
	<li>Lavar o útero todo com PBS 1x em uma placa de Petri.</li>
	<li>Corte o útero desenvolve com tesoura de dissecação e retire da placenta decídua com dissecando o fórceps para expor os embriões em gema sacs.</li>
	<li>Primeiro remover o saco vitelino e remova a membrana amniótica com dissecando o fórceps para libertar os embriões perinatais.</li>
	<li>Decapitar um embrião com tesoura de dissecação. Limpe o excesso de sangue com gaze estéril. Levanta o embrião para baixo com sua superfície ventral na dissecação microscópio equipado com uma câmera digital para a imagem latente. Recolher a sua cauda para a genotipagem, se necessário. 
	Nota: Executar um embrião de injeções de cada vez.</li>
	<li>Abra cuidadosamente a cavidade abdominal de um embrião com fórceps, rasgando a pele. Em seguida, remover cuidadosamente o excesso órgãos e tecidos como fígado, estômago e intestino com fórceps cortá-los ou puxá-los para fora para expor os rins, os ureteres e a bexiga, que está localizado dorsalmente ( figura 1A). Absorver o excesso de sangue do embrião dissecado com gaze estéril, se necessário. 
	Nota: Sangue excesso interfere com a identificação da pelve renal para a injeção do corante.</li>
</ol> 3. Injeção de corante azul de metileno na pelve Renal e monitorando o fluxo de tintura <ol><li>remover bolhas na agulha e tubulação, expulsando a solução de azul de metileno da ponta de agulha por pressão hidrostática. Levantar a seringa contendo a solução de tintura acima do nível da ponta da agulha para iniciar o fluxo e em seguida baixar a seringa para parar o fluxo.</li>
	<li>Insira a agulha na pelve renal perto do ureter proximal, tomando cuidado para não perturbá-lo depois de colocado. Realizar injeção de corante em um rim para determinar sua obstrução do tracto urinário. 
	Nota: Executar tingir a injeção em qualquer anormal do rim 7 primeiro, seguindo a mesma maneira para determinar sua obstrução do tracto urinário.</li>
	<li>Levantar a seringa até cerca de 20 cm para fornecer a pressão hidrostática e deixe 15 µ l - 60 µ l do fluxo de solução corante. A taxa de fluxo será cerca de 3 µ l/s, se a seringa é gerada para esta altura acima do embrião.</li>
	<li>Monitor a cor azul do corante começando primeiro na pelve renal e, em seguida, no comprimento do ureter e finalmente no lúmen da bexiga. 
	Nota: Demora cerca de 5 s para ver uma cor de tintura fraca emergem dentro do lúmen da bexiga se o ureter está correctamente inserido na bexiga. Permitir que a tintura a acumular-se no site bloqueado cerca de 15 s se nenhuma cor corante aparece no lúmen da bexiga.</li>
	<li>Coloque a seringa fluxo a tintura pare e retire a agulha do rim.</li>
	<li>Tomar imagens do rim, ureter e bexiga rastreados com solução corante usando a câmera e imagem programa conectem a um estereomicroscópio.</li>
	<li>Fazer um registro de hidronefrose do rim, hidroureter e a posição final da solução corante em um caderno de anotações.</li>
	<li>Realizar injeção do rim contralateral conforme descrito nas seções 3.1) para 3.5) e permitir o fluxo de tinta cerca de 20 s total. 
	Nota: O lúmen da bexiga será preenchido com solução corante, indicada por uma cor azul forte, se o ureter está correctamente inserido o lúmen da bexiga. Após a avaliação da obstrução de junção do tracto urinário, os ureteres e a bexiga podem ser corrigidos para secionar.</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>

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Rins de mouse são funcional início em E16.5 e um teste de injeção de corante é teoricamente possível deste ponto do tempo. No entanto, o rim é muito pequeno para ser injetado com a solução de corante e fenótipos como hidronefrose e hidroureter não são claramente observados desde que estes fenótipos são um efeito secundário da urina acumulada devido ao transporte de urina anormal. Estes fenótipos, desde as obstruções, como UPJO ou UVJO, são evidentes no E18.5 por distendido rins e ureteres. Maiores rins...

O sistema urinário consiste de um par de rins e ureteres e um comum da bexiga e uretra. A principal função do sistema urinário é manter a homeostase do corpo gerindo o equilíbrio de água e eletrólitos do sangue. Os rins filtram o sangue para controle concentrações de eletrólitos e equilíbrio ácido-base e produzem urina para excretar o excesso de água e resíduos, incluindo solutos e metabólitos. Urina é então transportada através do ureter da pelve renal do rim para o bladderin de uma maneira unidirecional, onde é armazenado e, finalmente, eliminada através da uretra1.
Os ureteres são tubos retos, proveniente do ducto nephric. Após a brotação do ducto nephric em dia embrionário 10.5 (E10.5) no mouse, o caule do uréter alonga e diferencia em uma estrutura de várias camada chamada Urotélio, que é impermeável entre rato, E12.5 e E16.5. As células mesenquimais em torno do caule de ureter também são diferenciadas em três camadas consistindo de células do estroma internas, células de músculo liso grosso e intermediário e fibroblastos adventícia exteriores. Ondas peristálticas ureterais iniciando na pelve renal são propagadas através da camada de músculo liso da parede da uretra à bexiga para transportar urina2,3, que é produzido começando em E16.5 no rato1.
Anomalias congênitas do rim e trato urinário (CAKUT) estão entre as mais frequentes doenças genéticas, presentes em cerca de 1% dos fetos humanos1,4e composto por uma variedade de fenótipos incluindo hidronefrose e hidroureter. O acúmulo anormal de urina nos resultados da rim e do ureter no rim e hidroureter formação de hydronephrotic. Uma das causas da formação de hidronefrose e/ou hidroureter é uma obstrução do tracto urinário. Obstrução de junção de ureteric (UPJO) é causada pelo fluxo de urina aberrante, devido a um bloqueio entre o ureter proximal e a pelve renal, resultando em hidronefrose e hidroureter proximal estreitando com angulação ou dobramento persistente5, 6. Além disso, a inserção ectópica do ureter distal na parede da bexiga ou do trato reprodutivo é chamada ureter obstrução de junção (UVJO). UVJO também pode induzir a hidronefrose e hydromegaureter formação7,8. Defeito de junção (como) um ureter adicional é refluxo vesicoureteral (RVU). VUR é caracterizada pelo fluxo retrógrado de urina da bexiga para o rim em como. Em comparação com UVJO, perinatais embriões com RVU não distintamente mostra um rim distendido hydronephrotic ou grave hidroureter fenótipo9.
No rato de laboratório, o fluxo de urina pode ser examinado por injeção de um corante, tais como o azul de metileno, para a pelve renal9. A solução de azul de injectedmethylene irá traçar a trajetória de urina da pelve renal, através da uretra e na bexiga. Hidronefrose pode ser reconhecido por uma expansão do corante no rim. UPJO pode ser detectado como uma obstrução do fluxo do corante para o ureter proximal com distendido pelve renal5. Qualquer dilatação do ureter indicado por um diâmetro ampliado demonstra um exemplo de hidroureter. Finalmente, acúmulo de tinta na parede da bexiga ou no site do tracto reprodutivo indica UVJO com rim hydronephrotic distendido e dilatada hydromegaureter7,10. Para detectar o RVU, a solução corante é injectada na bexiga e subsequente fluxo retrógrado é monitorado na renal9.
Aqui, um protocolo para injeção de corante azul de metileno em pelve renal de um embrião perinatal é apresentado. Este protocolo permite o rastreamento do fluxo de urina da pelve renal, através da uretra e na bexiga e verifica possíveis obstruções de junção do trato urinário como UPJO ou 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

Defeitos de obstrução do trato urinário junção são anomalias congênitas induzindo hidronefrose e hidroureter. Defeitos de obstrução urinária murino junção podem ser avaliados pelo controle de fluxo do corante azul de metileno dentro do sistema urinário. Corante azul de metileno é injetado em pelve renal dos rins embrionários perinatais e fluxo do corante é monitorado da pelve renal do rim através do ureter e para dentro do lúmen da bexiga após a aplicação de pressão hidrostática. Acumulação de tintura será evidente no lúmen do tracto urinário perinatal normal da bexiga, mas vai ser restrita entre a pelve renal e o ponto final de um ureter anormal, se ocorrerem obstruções do trato urinário. Esse método facilita a visualização de hidronefrose e hidroureter e a confirmação da obstrução de junção do trato urinário. Este manuscrito descreve um protocolo para injeção de corante azul de metileno na pelve renal para confirmar obstruções de junção do trato urinário.

Rins e sistema urinário inferior estão localizados dorsal à maioria dos outros órgãos internos como o fígado e intestino. Depois de remover esses outros órgãos internos, um par de rins e ureteres e bexiga única são visíveis (figura 1A). Após dissecação bem sucedida, o corante injetado em pelve renal fluirá do rim para a bexiga através da uretra. A pelve renal é a funil-como dilatada porção proximal do ureter no rim. Portanto, o ponto de inj...

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

Injeção de corante azul de metileno em pelve renal facilita a avaliação de defeitos de obstrução do trato urinário junção durante o desenvolvimento embrionário do trato urinário de rato. Aqui, um protocolo para injeção de corante azul de metileno na pelve renal é descrito.

Avaliar defeitos de obstrução do trato urinário junção por injeção de corante azul de metileno

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 visualizações.  National Institutes of Health. Injeção de corante azul de metileno em pelve renal facilita a avaliação de defeitos de obstrução do trato urinário junção durante o desenvolvimento embrionário do trato urinário de rato. Aqui, um protocolo para injeção de corante azul de metileno na pelve renal é descrito.

Vídeo: 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.

Injeção do mouse parede da bexiga

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

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).

A aplicação de 1% de corante azul de metileno como uma única técnica na biópsia do nó Sentinela do cancro da mama

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 ...

Pesquisa do câncer

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 fotoacústica

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

Bioengenharia

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.

Intraoperatória Detecção de Endometriose Sutil: um novo paradigma para a detecção e tratamento de dor pélvica associada com a perda de integridade Peritoneal

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.

Sonda baseada Endomicroscopy Laser Confocal do Trato Urinário: A Técnica

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.

Procedimento de instilação transuretral no rato adulto masculino

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

Imunologia e Infecção

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).

Ultrassonografia do trato urinário masculino adulto para testes funcionais urinários

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.

Usando a microscopia de 2-fótons para quantificar os efeitos da obstrução ureteral unilateral crônica nos processos glomerulares

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 Medição Luminal da Liberação de ATP Urotelial Evocado por Distensão em Roedores

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.

Um modelo murino imaturo de obstrução ureteral unilateral reversível

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