Anesthesia used: Ketamine / Xylazine, Isoflurane 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 s terile 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. All glass pipettes used are soaked in 70% ethanol then wiped dry with a sterile gauze or paper towel.
Preparation of the animal: The animal was injected IP with Ketamine and maintained if necessary with Isoflurane. Ophthalmic ointment is then placed on the animal’s eyes to prevent them from drying out during surgery. The animal’s hair is clipped on the abdomen side starting from the bottom of the rib cage down to just above the penis sheath and from each side of the abdomen. 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 for the procedure.
Monitoring: After the IP Ketamine injection to the animal, it is determined fully anesthetized when it lose its reflexes. A nose cone containing 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: With a pair of fine forceps, lift an area of skin 2 mm above the prepucial gland bulge, about 1-2 cm above the penis sheath, and about 2-3 cm below the bottom of the rib cage, make a horizontal incision 1 cm in length through the skin using surgical scissors. Then make the same type of incision through the muscle layer. Once through the body cavity, the bladder should be visible. With a pair of fine forceps grip the bladder and lift upward then downward out of the body cavity towards the penis sheath. This will expose the two seminal vesicles. While holding onto the bladder with your left hand, gently pull out of the body cavity the animal’s left seminal vesicle so that the seminal vesicle is lying downward, facing the animal’s penis sheath. A 27-gauge syringe needle is inserted into the large tubule of the left anterior prostate lobe, from the tip of the prostate lobe to its point of insertion. Doing so makes a pocket-like tunnel through the tubule, where the recombinant drop will be placed. Once, the pocket is made, the tissue recombinant will be inserted into the pocket with a homemade glass pipette. The glass pipette has a tip on the end that guides the recombinant into the tunnel. Once the glass pipette has guided the recombinant into the anterior prostate, near the point of insertion, use forceps to clamp over the pipette as the pipette is pulled out of the prostate lobe. This helps the recombinant stay in place as the pipette is removed. A suture can be used if the recombinant does not stay in place. Carefully lift the seminal vesicle with your fine forceps and insert it back into the body cavity, then the bladder. Once the organs are placed back into the body cavity, the muscle layer is sutured at least three times with 4-0 gut sutures with a reverse cutting, 3/8 circle C-13 needle. Once sutured, with forceps pull together the skin and clamp the skin closed with surgical clamps, closing the incision completely. The procedure is now completed. 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 recumbancy and is walking around the cage.