Animals were obtained from our own mouse colony housed in our campus’ animal facility at a weght rangingi 17-28 grams.
Anesthesia used: Ketamine / Xylazine, Isolflurane as needed.
Preparation of the surgical area: The surgical area that was used was aseptically sterilized, had proper lighting and contained with the appropriate surgical equipment. This room is designated as the surgery room for all rodent surgeries. An aseptically cleaned dissecting microscope is used to perform the surgical procedure. Sterile paper towels are placed on the stage of the microscope and the animal is then placed on top of the paper towel, backside down, and surgical area facing upward. The microscope also has added lighting to the surgical area. The surgical room is set up with appropriate heating pads that are used during the surgical procedure and a sterile blue pad was placed on top of the heating pads. A heating pad, with a sterile blue pad on top of it, is also used to place the rodents on during the post-operative procedures and while they are monitored. A sterile blue pad is also placed on the surgery-operating table next to the microscope for placement of the surgical instruments and supplies that are necessary for the surgical procedure.
Preparation of surgical instruments and supplies: All of the instruments and supplies (i.e. tools, gauze, staples, etc.) are sterilized by autoclave. A homemade glass pipette that is needed is soaked in 70% ethanol then wiped dry with a sterile paper towel or sterile gauze.
Preparation of the animal: The animal was injected IP with Ketamine and maintained if necessary with Isoflurane. Ophthalmic ointment was then placed on the animal’s eyes to prevent them from drying out during surgery. The animal’s hair is shaved 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. A small patch of hair is also shaved on the back only if a dihyrotestosterone pellet is implanted. The shaved area(s) are then prepped with betadine scrub, rubbing in a circular motion. 70% alcohol is then applied to the surgical area to remove the betadine and loose hair. This process is repeated 2 or more times. The betadine and alcohol is applied with sterile gauze. Once the animal is prepped for surgery, it is then laid on its back on the sterile paper towels that lay on top of the stage of the microscope and is now ready for the procedure.
Preparation for the surgeon: The surgeon wears a facemask, hair bonnet, surgical gown and clean, sterile foot covers. Before beginning the surgery, the surgeon thoroughly washes her hands and puts on a pair of sterile gloves. Once the surgeon is properly gowned and gloved, she places her sterile instruments on the sterile blue pad next to the microscope as well as sterile sutures and other necessary supplies.
Monitoring: After the IP Ketamine injection to the animal, it is determined fully anesthetized when it lose its reflexes. A nose cone contained isoflurane is used only if needed during the surgical procedure. During preparation and surgery, the anesceptic depth is determined by the animal’s respiratory pattern and by pinching the animal’s foot for reflex response. After the post-operative procedures, the animal is placed into a clean cage, which is placed on top of a heating pad at the appropriate temperature, lined with a blue pad. The animal is monitored until it has awakened from the procedure and is moving around the cage normally. This usually takes 30 minutes to one hour. The animal is then observed on a daily basis by the surgeon until sacrificed.
Analgesia: The animal is given a subcutaneous injection of Banamine after the procedure is finished. The animal is also given a subcutaneous injection of room temperature sterile saline directly after the surgery is finished, prior to waking up.
Operative description: The mouse is placed on its abdomen on the sterile paper towels that cover the stage of the microscope. A vertical incision is made through the skin along the animal’s spine, about 2 cm from the base of the tail to the top curve of the spine. The animal is laid on its left side. The surgeon’s right thumb is placed on the animal’s abdomen, applying some pressure and with the left hand the skin is pulled down, so that the kidney is in view, once the kidney is found, a .5cm incision is horizontally made on the animal’s right side with surgical scissors. With the aid of the surgeon’s thumbs and a little pressure on the abdomen, the kidney is exposed and popped out of the body cavity. If the incision is made too large, then the kidney will not stay popped out. This incision is critical is the success of this procedure. A small tear with a pair of sharp fine forceps is made in the kidney capsule. With your left hand, hold and gently pull up, so that the glass pipette can be inserted just beneath the kidney capsule and the top of the kidney. Be careful not to puncture the kidney. Move the glass pipette in a circular motion, making a small pocket for the recombinant to be placed. Guide the recombinant into the pocket, under the kidney capsule. Push the recombinant as far in as possible, so that it stays in place and does not leak out. The kidney is now ready to go back into 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, using 4-0 gut suture with a reverse cutting, 3/8 circle C-13 needle.. 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. This completes the procedure. Upon completion of the surgery, the animal is placed on a warm heating pad, covered with a blue pad, to aid in regaining its body temperature. At this time, Banamine, an analgesic, is administered at .04cc subcutaneously in the right flank. One cc of sterile saline is given subcutaneously directly behind the neck. Observe the animal until he has regained full sternal recumbence and is walking around the cage.