Methylene Blue Dye Injection in Mouse Embryonic Urinary Tract: A Method To Assess the Congenital Obstruction in the Urinary Tract

To assess urinary tract obstruction, begin by immobilizing a perinatal mouse embryo in the supine position. Now, dissect the abdominal cavity of the embryo to locate a fully developed urinary tract, which consists of a pair of kidneys, ureters, and a common bladder with urethra.

Next, take an empty syringe connected to the tubing fitted with a microneedle. Fill the assembly with methylene blue solution - a deep blue tracking dye. Insert the microneedle into the lumen of the renal pelvis - a funnel-like dilated proximal end of the ureter in the kidney - and inject the dye solution.

Due to hydrostatic pressure, the dye flows from the renal pelvis of the kidneys through the ureter and finally accumulates into the lumen of the bladder. Repeat the dye injection procedure with the contralateral kidney to check the flow of dye from the corresponding side of the urinary tract.

Finally, use a stereomicroscope with a camera to image the urinary tract. Upon imaging, an unobstructed urinary tract shows a deeply stained bladder, while the abnormal urinary tract with obstruction restricts the free flow of the dye. Depending on the degree of blockage, a complete or partial absence of dye in the bladder lumen is observed.

Using forceps, carefully tear the skin to open the abdominal cavity of the embryo. Then, carefully remove excess organs and tissues such as liver, stomach, and intestines with forceps by cutting them or pulling them out to expose the kidneys, the ureters, and the bladder that are located dorsally. Absorb excess blood from the dissected embryo with sterile gauze pads, then continue to dye injection.

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. Next, 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.

Appropriate insertion of a needle into the renal pelvis determines the success of this test. A needle should be inserted into the renal pelvis toward the ureter, neither passing through the kidney nor toward the blood vessel.

Lift the syringe up about 20 centimeters to provide hydrostatic pressure and let 15 to 16 microliters of the dye solution flow. Then, 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. Place the syringe down to stop the dye flow and remove the needle from the kidney. Make a record of hydronephrosis of the kidney, hydroureter, and the final position of the dye solution in a lab notebook.

Finally, perform injection of the contralateral kidney and allow the dye to flow for 20 seconds total. Take images of the kidney, the ureter, and the bladder traced with dye solution using the camera and imaging program connected to a stereomicroscope.