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

I thank Dr. Alan O. Perantoni (CDBL/NCI/NIH) for supporting the submission of this manuscript. I also thank Dr. Michael Hall (CDBL/NCI/NIH) and Nirmala Sharma (CDBL/NCI/NIH) for editing this manuscript. I am grateful to Lai Thang (SAIC) for his excellent mouse husbandry. This work was supported by the National Institutes of Health, National Cancer Institute and Center for Cancer Research.

Mice (Wnt5a flox/flox mice (Wnt5atm1.1Tpy) and Dll1Cre line, UVJO mouse model)7 were managed according to NIH guidelines for the care and use of laboratory animals and studied under a protocol approved by the NCI-Frederick Animal Care and Use Committee.
1. Preparation of Methylene Blue Dye Solution
<ol>
	<li>Measure 0.1 g methylene blue powder.</li>
	<li>Dissolve the methylene blue in 10 mL normal saline or 1x phosphate buffered saline (PBS) completely by vortexing.</li>
	<li>Filter the 10 mg/mL methylene blue solution with a syringe filter (membrane pore size 0.45&#181;m) to eliminate clogging during injection.</li>
	<li>Assemble a sterile scalp vein set (27GX3/4&#8243;) with a 3 mL disposable syringe and fill the syringe with 3 mL filtered methylene blue solution. 
	NOTE: To prevent hydrostatic pressure and subsequent dye flow, place the needle tip above the syringe filled with methylene blue solution.</li>
</ol>
2. Dissection of Prenatal Embryos
<ol>
	<li>Clean dissecting scissors and forceps with 70% ethanol.</li>
	<li>To collect perinatal embryos at E18.5 or E19.5, euthanize pregnant mice first using CO2 inhalation and then perform cervical dislocation according to NIH guidelines. 
	NOTE: The pregnant female mouse usually gives birth starting at E18.5, however, larger kidneys are easier to manipulate. Therefore, kidneys at E19.5 closer to birth are easier to perform this analysis on. However, pups with bilateral urinary tract obstructions die after birth. Depending on the characteristics of experimental mice, an appropriate collection day/time should be empirically determined.</li>
	<li>Spray 70% ethanol on the ventral abdominal surface and then open the abdominal cavity ventrally using dissecting scissors and forceps.</li>
	<li>Lift the entire uterus and separate it from the body by cutting with dissecting scissors at the tips of the uterine horns.</li>
	<li>Rinse the entire uterus with 1x PBS in a petri dish.</li>
	<li>Cut the uterus segmentally with dissecting scissors and remove placental decidua with dissecting forceps to expose the embryos in yolk sacs.</li>
	<li>Remove the yolk sac first and then remove the amniotic membrane with dissecting forceps to liberate the perinatal embryos.</li>
	<li>Decapitate an embryo with dissecting scissors. Wipe off excess blood with sterile gauze pads. Pin the embryo down with its ventral surface up on a dissecting microscope equipped with a digital camera for imaging. Collect its tail for genotyping if necessary. 
	NOTE: Perform injections one embryo at a time.</li>
	<li>Carefully open the abdominal cavity of an embryo with forceps by tearing the skin. Then, carefully remove excess organs and tissues such as liver, stomach, and intestine with forceps by cutting them or pulling them out to expose the kidneys, the ureters, and the bladder that are located dorsally (Figure 1A). Absorb excess blood from the dissected embryo with sterile gauze pads if necessary. 
	NOTE: Excess blood interferes with identification of the renal pelvis for the dye injection.</li>
</ol>
3. Injection of Methylene Blue Dye into the Renal Pelvis and Monitoring Dye Flow
<ol>
	<li>Remove bubbles in the needle and tubing by expelling methylene blue solution from the needle tip by hydrostatic pressure. Lift the syringe containing the dye solution above the level of the needle tip to start flow, and then lower the syringe to stop flow.</li>
	<li>Insert the needle into the renal pelvis near the proximal ureter, taking care not to disturb it once placed. Perform dye injection into a kidney to determine its urinary tract obstruction. 
	NOTE: Perform dye injection into any abnormal kidney7 first by following the same way to determine its urinary tract obstruction.</li>
	<li>Lift the syringe up about 20 cm to provide hydrostatic pressure and let 15 &#181;L - 60 &#181;L of the dye solution flow. The flow rate will be about 3 &#181;L/s if the syringe is raised to this height above the embryo.</li>
	<li>Monitor the blue color of the dye starting first in the renal pelvis, then in the length of the ureter, and finally in the bladder lumen. 
	NOTE: It takes about 5 s to see a weak dye color emerge within the bladder lumen if the ureter is properly inserted into the bladder. Allow the dye to accumulate at the blocked site about 15 s if no dye color appears in the bladder lumen.</li>
	<li>Place the syringe down to stop dye flow and remove the needle from the kidney.</li>
	<li>Take images of the kidney, the ureter, and the bladder traced with dye solution using the camera and imaging program connected to a stereomicroscope.</li>
	<li>Make a record of hydronephrosis of the kidney, hydroureter, and the final position of the dye solution in a lab notebook.</li>
	<li>Perform injection of the contralateral kidney as described in sections 3.1) to 3.5) and allow dye flow about 20 s in total. 
	NOTE: The bladder lumen will be filled with dye solution, indicated by a strong blue color, if the ureter is properly inserted into the bladder lumen. After assessment of urinary tract junction obstruction, the ureters and the bladder can be fixed for sectioning.</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>

The author has nothing to disclose.

Mouse kidneys are functional beginning at E16.5 and a dye injection test is theoretically possible from this time point. However, the kidney is too small to be injected with the dye solution and phenotypes such as hydronephrosis and hydroureter are not clearly observed since these phenotypes are a secondary effect of urine built up due to abnormal urine transport. These phenotypes, from the obstructions such as UPJO or UVJO, are evident at E18.5 by distended kidneys and ureters. Larger kidneys of embryos at E19.5 or neonatal pups are easier to manipulate than those of embryos at E18.5 or younger. However, pups with bilateral urinary tract obstructions die after birth. Therefore, perform experiments to validate CAKUT phenotypes by appropriately choosing perinatal embryos or pups depending on experimental mouse characteristics.
Dissect carefully to maintain an intact urinary system prior to performing dye injection to examine potential defects. This is essentially important for the abnormal hydronephrotic kidneys and ureters, which are very distended and easily damaged during the removal of extra tissue to expose the kidneys and urinary tracts. Damaging these organs during dissection will prevent a full determination of real defects. For example, Figure 2 shows distinct dilation of a ureter, but the dye leakage fails to show the terminal point of the distal hydroureter.
Always be sure to remove air bubbles from the tubing and the needle prior to injection, as these bubbles will block proper dye flow. After successful dissection, the critical step to examine the urinary tract obstruction is insertion of the needle tip into the renal pelvis. The insertion of the needle into the upper half of the kidney, near the proximal ureter, is helpful to direct the needle tip into the renal pelvis. The needle tip should point toward the proximal ureter. Since the renal blood vessels are located near the proximal ureter, the dye may flow along the renal blood vessels unless the needle tip points toward the proximal ureter. In this case, the needle tip should be adjusted in the renal pelvis toward the proximal ureter. Further, the needle may pass through the kidney if inserted too deep, causing the dye to leak out of the kidney and into the body cavity. Careful insertion of the needle tip into the renal pelvis and monitoring the needle tip within the renal pelvis can help avoid this failure. Sometimes, the needle will become blocked by kidney tissue during injection if it is not inserted into the renal pelvis. In this case, remove the tissue from the needle, reset dye flow by restarting hydrostatic pressure and redirect the needle tip toward the renal pelvis. In addition, phenotypes associated with CAKUT may be unilateral or bilateral. Therefore, ensure that the injection is performed separately for left and right urinary systems. Optimally, perform the injection first with an obviously abnormal kidney to verify whether UVJO is unilateral.
Image immediately after the injection test since the dye passes from the renal pelvis through the ureter quickly. It is hard to see the color during imaging of a normal ureter because a normal ureter is a thin tube, though dye flow can be monitored under the dissecting microscope. As an alternate to methylene blue, 1% fast green in 1x PBS can also be used and will appear as a dark green color9. In addition to dye injection, histological sections of the kidneys and ureters following H&#38;E staining will confirm the defects within the kidneys and ureters. Also, imaging of sections with a ureter specific reporter is an additional way to confirm UVJO7. However, these sections should be intact and serial sectioning is necessary to avoid false positives. Compared with traditional section-based analyses of urinary systems, this dye injection protocol is an instant way to observe the integrity of the kidney and patent ureter and to visualize obstructions such as UPJO or UVJO.
This protocol provides a simple and straightforward method toquickly visualize urinary tract structure in late stage mouse development, which allows the analysis of urinary tract development. The method described here can be used to assess mutations that cause developmental urinary tract defects, as a means of identifying changes in urinary tract maturation or ureter development.

The urinary system consists of a pair of kidneys and ureters and a common bladder and urethra. The main function of the urinary system is to maintain body homeostasis by managing the water and electrolyte balance of the blood. The kidneys filter the blood to control electrolyte concentrations and acid-base balance,and produce urine to excrete excess water and waste including solutes and metabolites. Urine is then transported through the ureter from the renal pelvis of the kidney to the bladderin a unidirectional manner where it is stored and ultimately eliminated via the urethra1.
The ureters are straight tubes originating from the nephric duct. After budding from the nephric duct at embryonic day 10.5 (E10.5) in the mouse, the ureter stalk elongates and differentiates into a multilayered structure called urothelium which is impermeable between mouse E12.5 and E16.5. The mesenchymal cells surrounding the ureter stalk are also differentiated into three layers consisting of inner stromal cells, intermediate thick smooth muscle cells, and outer adventitial fibroblasts. Ureteral peristaltic waves initiating in the renal pelvis are propagated through the smooth muscle layer of the ureter wall to the bladder to transport urine2,3, which is produced starting at E16.5 in the mouse1.
Congenital anomalies of the kidney and urinary tract (CAKUT) are among the most frequent genetic diseases, present in around 1% of human fetuses1,4, and composed of a variety of phenotypes including hydronephrosis and hydroureter. The abnormal accumulation of urine in the kidney and ureter results in hydronephrotic kidney and hydroureter formation. One cause of hydronephrosis and/or hydroureter formation is an obstruction of the urinary tract. Ureteropelvic junction obstruction (UPJO) is caused by aberrant urine flow due to a blockage between the proximal ureter and the renal pelvis, resulting in hydronephrosis and proximal hydroureter narrowing with angulation or persistent folding5,6. In addition, the ectopic insertion of the distal ureter into either the bladder wall or the reproductive tract is called ureterovesical junction obstruction (UVJO). UVJO can also induce hydronephrosis and hydromegaureter formation7,8. An additional ureterovesical junction (UVJ) defect is vesicoureteric reflux (VUR). VUR is characterized by retrograde urine flow from the bladder toward the kidney at UVJ. Compared to UVJO, perinatal embryos with VUR do not distinctly show a distended hydronephrotic kidney or severe hydroureter phenotype9.
In the laboratory mouse, urine flow can be examined by injection of a dye, such as methylene blue, into the renal pelvis9. The injectedmethylene blue solution will trace the urine trajectory from the renal pelvis through the ureter and into the bladder. Hydronephrosis can be recognized by an expansion of the dye in the kidney. UPJO can be detected as a blockage of dye flow at the proximal ureter with distended renal pelvis5. Any dilation of the ureter indicated by a widened diameter demonstrates an example of hydroureter. Finally, dye accumulation at the bladder wall or at the site of the reproductive tract indicates UVJO with distended hydronephrotic kidney and dilated hydromegaureter7,10. To detect VUR, the dye solution is injected into the bladder and subsequent retrograde flow is monitored in the kidney9.
Here, a protocol for methylene blue dye injection into the renal pelvis of a perinatal embryo is presented. This protocol allows the tracing of urine flow from the renal pelvis through the ureter and into the bladder and verifies potential urinary tract junction obstructions such as UPJO or 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

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction defects can be assessed by tracking methylene blue dye flow within the urinary system. Methylene blue dye is injected into the renal pelvis of perinatal embryonic kidneys and dye flow is monitored from the renal pelvis of the kidney through the ureter and into the bladder lumen after applying hydrostatic pressure. Dye accumulation will be evident in the bladder lumen of the normal perinatal urinary tract, but will be constrained between the renal pelvis and the end point of an abnormal ureter, if urinary tract obstructions occur. This method facilitates the confirmation of urinary tract junction obstructions and visualization of hydronephrosis and hydroureter. This manuscript describes a protocol for methylene blue dye injection into the renal pelvis to confirm urinary tract junction obstructions.

The kidneys and lower urinary system are located dorsal to most other internal organs such as the liver and intestine. After removing these other internal organs, a pair of kidneys and ureters and a single bladder are visible (Figure 1A). Upon successful dissection, the dye injected into the renal pelvis will flow from the kidney into the bladder via the ureter. The renal pelvis is the funnel-like dilated proximal part of the ureter in the kidney. Therefore, the dye injection point is near the visible proximal ureter connected with the kidney as indicated with black arrows (Figure 1B-1C). After injection with methylene blue dye solution into one kidney, the dye flows from the renal pelvis through the ureter and into the bladder resulting in a visible blue color in the ureter and bladder lumen, if urinary tract obstructions do not exist (Figure 1B). The dye solution usually disappears in a normal ureter quickly even before imaging, and the dye color in the ureter is weak because the normal ureter is a thin tube. However, dye flow in the ureter can be observed with a dissecting microscope. A strong blue color in the bladder lumen appears as the result of a longer flow of about 20 s in total after injection of both kidneys with the dye solution (Figure 1C). Dye accumulation in the bladder after the contralateral injection indicates that the second urinary system does not have any urinary tract junction obstruction.
It is reasonable to inject an obviously abnormal kidney first to confirm any urinary tract junction obstructions. Abnormal hydronephrotic kidneys can be recognized by their bigger size or thin transparent surface with a broadened renal pelvis. An example distended hydronephrotic kidney and an abnormally dilated hydroureter are depicted in Figure 2A. A dilated hydroureter usually results in a much stronger visualization because its expanded volume retains much more dye solution than a normal, thin ureter. If the kidneys and/or the ureters are damaged during dissection, the examination of urinary tract junction obstructions is impossible because the dye will leak out aberrantly at the damaged site. For example, the abnormal hydroureter in Figure 2A was damaged in its middle and, therefore, the final position of the dye solution is unclear and UVJO can&#39;t be verified despite the absence of the dye in the bladder lumen (Figure 2A). However, this perinatal urinary system with dilated hydroureter and distended hydronephrotic kidney may have UVJO as the ureter is widened along its length and the kidney is distinctly distended. The contralateral urinary system appears normal as it has a thin ureter and the dye subsequently enters the bladder lumen (Figure 2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56247/56247fig1.jpg" /> 
Figure 1. Examples of methylene blue dye injection into a normal urinary system. (A) A typical image of the urinary system from a perinatal embryo (E19.5) after removing excess tissue. Two kidneys, two ureters, and a bladder are seen with reproductive organs, here testes (B) An example of dye injection into a normal kidney. An injection point into the renal pelvis of the kidney (black arrow) can be seen. A weak dye color is visible in the ureter and the bladder lumen after allowing the dye to flow for 5 s. (C) Dye accumulation in the bladder lumen after injection of both normal kidneys. The color becomes strong from the accumulation of the dye solution in the bladder lumen after allowing the dye to flow for 20 s. The renal pelvis is also visible with the weak dye color in the kidney. B: bladder, K: kidney, T: testis, U: ureter. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56247/56247fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56247/56247fig2.jpg" /> 
Figure 2. Examples of methylene blue dye injection into a urinary system with unilateral hydroureter. (A) An example of dye injection into a urinary system with a hydroureter and a hydronephrotic kidney of a neonatal female pup with dye flow of 15 s. The hydronephrotic kidney is clearly bigger than the contralateral kidney. The corresponding ureter is dilated and the dye color is strongly recognizable but the hydroureter is clearly damaged as observed by leaking dye solution in the body cavity. (B) Dye injection into the contralateral kidney of a urinary system with unilateral hydroureter with dye flow of 20 s. Injected dye solution into the normal kidney accumulates in the bladder lumen confirming that the abnormality is unilateral. B: bladder, HK: hydronephrotic kidney, HU: hydroureter K: kidney, U: ureter, Ut: uterine horn. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56247/56247fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

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 San Sebastian

Research

JoVE Journal

Biology

15.6K Views.  National Institutes of Health. Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

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.

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

Medicine

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).

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 ...

Cancer Research

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.

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

Bioengineering

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.

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.

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  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.

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

Immunology and Infection

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).

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.

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.

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