Urine Depletion and Bladder Rinsing in Mouse Model: A Catheter-based Procedure to Empty and Wash Murine Urinary Bladder

протокол

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

 1. Anesthetizing Animal

1. Use a tabletop anesthesia system to anesthetize the animal with isoflurane. Turn on the O₂ tank and adjust the oxygen flowmeter to 1 L/min.
2. Place an adult female C57BL/6J mouse in the induction chamber. Turn on and adjust the isoflurane vaporizer to 3% for induction. The mouse should be in anesthesia within 2 min. Administer buprenorphine analgesic (0.1 mg/kg) subcutaneously upon removal of the mouse from the induction chamber for preemptive analgesia.
   NOTE: In this protocol, female mice are used, as they are easier to work with during urethra catheterization. The protocol also works for male mice, but extra caution is needed.
3. Apply ophthalmic ointment to the eyes of the animal to prevent dryness. Place the anesthetized mouse facing up on a heating pad with its nose in the nose cone. Turn the air circuit switch to let isoflurane go through the nose cone.
4. Restrain the head and the nose cone with adhesive tape. Adjust the isoflurane vaporizer to 2% for maintenance.
5. Restrain the limbs with adhesive tape. Perform toe-pinch tests to make sure the animal is in deep anesthesia and then shave the abdominal area.

2. Urine Depletion and Bladder Rinsing

1. Apply lubricant to the catheter (24G, length of 2.11 cm, outer diameter of 0.045 cm) and ensure that its outer surface is fully covered.
2. Insert the catheter into the urethra opening and slowly push it until it reaches the bladder, at which time the urine should flow out through the catheter. Gently press the abdomen to help urine depletion.
   1. Check if the catheter is inserted correctly into the bladder by observing automatic urine out-flow. Avoid piercing the urethra and bladder wall and apply lubricant multiple times if needed.
3. Discard the urine with a pipette into a waste beaker.
4. Pipette 80 µL of phosphate-buffered saline (PBS) into the outer end of the catheter, with the other end still in the bladder. Carefully attach a 1 mL syringe to the catheter. Gently push the syringe to inject the PBS into the bladder. Leave some PBS in the catheter to avoid creating air bubbles in the bladder.
5. Remove the syringe. Wait for PBS to drain out of the bladder. Gently press the abdomen to evacuate PBS.
6. Discard the PBS in the catheter with a pipette into the waste beaker.
7. Repeat PBS washing (steps 2.4 to 2.6) two more times.
   NOTE: It is essential to work gently during these washing steps. Blood in the catheter or failure of automatic out-flow of liquid usually indicates the urethra is pierced through

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Материалы

Name	Company	Catalog Number	Comments
BD 1mL TB Syringe	Fisher	148232E
Exel International Disposable Safelet I.V. Catheters	Fisher Scientific	14-841-21	24G, 2.11 cm length, 0.045 cm outer diameter
K&H Heated Resting Mat for Small Animals, 9 By 12 Inches	Amazon		Cover the heat mat with paper towels
Ophthalmic ointment	Fisher	NC0849514
PBS, pH7.4	Life Technologies	10010049
Pipet tips 200 µL	USA Scientific	1111-0206
Ophthalmic ointment	Fisher	NC0849514
Pipet Tips, Universal Fit; 0.1 to 10 µL	Fisher	02707438(CS)
Buprenorphine	Sigma	B9275-50MG
PIPETMAN NEO P2	Gilson	F144561
PIPETMAN NEO P200N	Gilson	F144565
Therio-gel Veterinary Lubricant	PBS animal health	353-736
V-1 Table top system anesthesia	VetEquip	901806
Isoflurane	Vet one	501017
Tape, Scotch	Fisher	19047257