Current Users of Procedure: Yan Xing

Hazards:

Ketamine and Buprenorphine are controlled substances and anyone working with these needs to take the training session and be registered the SBOP (State Board of Pharmacy). Go to this website to register for the training https://onestop2.umn.edu/training/courseDetail.jsp?course=RA0001

EXPOSURE TO ANIMALS: Personnel involved with animals may be exposed to bites, scratches, infectious agents, and allergens.

TETANOUS: a rare but potentially fatal bacterial infection is possible after any animal bite. See the Boynton Health Service brochures on animal bites and allergies in this manual.

CARBON DIOXIDE: toxic gas, avoid inhaling.

SHARPS: take appropriate cautions when working with needles

Protection:

During the surgery, surgeon should wear lab coat, gloves, face mask, and hair net.

Have current tetanus immunization.

If biten by a mouse, immediately wash wound with soap and water. Seek medical assistance from Boynton Health Service, if necessary (625-7900).

Waste:

Animal carcasses must be frozen and disposed of by Research Animal Resources. There is a freezer on the 2nd floor of Nils Hasselmo Hall.

Needles must be disposed of in a biohazard sharps container.

Spill clean-up: Isolate the area. Use appropriate tools to put materials into a convenient waste container.

Procedure:

Prepare reagents/tools:

-Have organ or cell preparation ready for transplant

-Prepare transplant needle: Remove insert from a spinal needle and trim with wire cutters to be ~1.5in or enough to match up with length of 18G/1.5in needle. File any rough edges to be flat and flush. This piece should fit and slide smoothly inside the 18G as it will be used to push the fetal thymus through the needle and under the capsule.

-Ketamine xylazine mix: load 120ul/mouse into a 1cc syringe

-Buprenorphine: dilute stock 1:10 in PBS, load 30ul/mouse into a 0.3 cc syringe

-Eye moisture salve, 70% EtOH, instruments (2 fine curved forceps, 1 fine scissors, 1 small scissors, cotton swabs), stapler with staples, sterile PBS dropper, Novalsan surgical solution, 4-0 gut sutures

- Sterilize hood with bleach, place a sterile blue pad, secure proper lighting and dip all surgical equipment in a 1:10 solution of Novalsan surgical scrub.

1. Anesthesia: Inject animals with 80ul ketamine/xylazine mix i.p. (use 1 cc tuberculin syringe, with 28g needle)

Note: as this anesthetic is a controlled substance, the lab stock is kept in a locked box under Jie’s bench. Code is available to those who have completed controlled substance training. From commercial stocks of 100 mg/ml Ketmine and 100 mg/ml xylazine, technicians made stocks that are 18 mg/ml ketamine and 1.8 mg/ml xylazine (10ml ketamine + 1ml xylazine into 44ml PBS). These are in 15 ml conical tubes. The precise amount used should be recorded in the Disposition Log in the Controlled substances notebook above Jie’s desk.

We are approved to inject 100ul of this i.p., which is 90 mg/kg ketamine for the average mouse (25 g). RAR recommends 25-75 mg/kg, so use 80ul to start. Anesthetic depth can be assessed prior to the procedure by pinching the toe. If after 5 minutes, the toe reflex is still active, the animal is not completely under, and a small amount more can be injected.

2. Surgery:

Apply eye moisture salve to the eyes of each animal to prevent them from drying out during the anesthesia period.

If necessary, shave animal’s hair on the dorsal side starting from about 2 cm above the animal’s tail along the spine to mid back and on each side of spine about 1 cm in width.

Prep incision area with Novalsan surgical scrub, using a sterile gauze pad rubbing in a circular motion. Repeat 2 or more times. Once the animal is prepped for surgery, lay it on its side with prepared side facing up on top of the on the blue pad.

Place the mouse on its side and make a skin incision approximately 1.5cm long in a longitudinal direction between the last rib and hip joint. Loosen connective tissue under the skin using fine forceps. Make a 0.5 cm incision in a longitudinal direction in the abdominal wall. Gently squeeze the kidney out onto the exterior surface of the abdominal wall and keep moist with a small amount of sterile PBS dropped onto it using a sterile dropper.

Load the fetal thymus onto the needle so it is just inside the tip. Perforate the kidney capsule using fine forceps, making a 1-2 mm hole, and being extremely careful not to puncture the kidney. Insert and guide the needle through the hole, under the kidney capsule, but above the kidney parenchyma. Once the needle is in place, gently push the internal piece (ie, spinal needle portion) to implant the fetal thymus. Slowly remove needle from under kidney capsule.

Return kidney to the body cavity: With a pair of fine forceps, hold onto the muscle layer surrounding the kidney and pull up all around the kidney, careful not to damage the kidney. The kidney will fall back into place into the body cavity. Once the kidney is in the body cavity, the muscle layer is sutured (2 sutures), using 4-0 gut sutures. Pull the skin layer upward and clamp the skin together using sterile surgical clamps, making sure that all of the skin is closed together and no gaps are left. Staple skin together with 2-3 staples. This completes the procedure.

3. Post-operative analgesia and monitoring

Lay the animal on its side in a clean cage on a heating pad sent on low to recover (may take up to 2 hours for complete recovery). Inject the analgesic buprenorphine (Buprenex). Dilute a .3mg/ml stock 1:10 in PBS, inject 30 ul of this (900ug) i.p .

Observe the animals until they have regained consciousness and are walking around the cage.

Further observe the animals daily for three days post procedure. Insure that the animals are active, eating, and drinking, and that the wound is healed. It will be assumed that all animals are healthy post-surgery unless indicated otherwise in the surgeon's notebook. NOTE: surgeon’s notebook indicating individual animal numbers and surgery/post-surgery evaluation dates MUST BE AVAILABLE TO INSPECTORS.

Include the following “checklist” in your notebook:

Anesthesia depth checked via toe-pinch

Ophthalmic ointment used

time of surgery ______

time of post-op assessment

1^st day _____

2^nd day______

2^nd day_____

3^rd day_____