To begin, place the sedated ambystoma Mexicanum with the ventral side down on wet paper towels soaked in 0.03%Benzocaine solution. Cover the animal with benzocaine-soaked paper towels. Use a pair of ring forceps to stretch out the operative hind limb.
With a scalpel, make a lateral longitudinal incision above the femur bone spanning the whole thigh in the upper hind limb. Carefully displace the muscles and nerves from the surgery site without cutting. Gently put Bode forceps under the femur to expose it for the surgery.
Now place a rigid 7.75 millimeter four-hole fixator plate along the femur diaphysis without touching the joints and secure it in the aligned position with forceps. Use four two-millimeter titanium screws to attach the bone to the plate. Under irrigation, with 0.7 times PBS, and 1%penicillin streptomycin, manually drill to create the first hole in the bone for screw insertion.
Then place the first screw in the hole. Apply the saw-guiding device onto the first screw and align it with the bone and plate. Use the saw-guiding device for drilling and inserting the remaining screws.
After ensuring alignment of the plate with the bone, remove the saw guide, and use a screwdriver to break off the handles from the screws. Place a piece of sterilized plastic film under the femur to prevent soft-tissue damage during the osteotomy process. Then place a Gigli wire saw between the bone and protection film.
Cut the bone using a Gigli wire saw creating a single 0.7 millimeter cut in the femur. Remove the saw and the protection film and irrigate the surgery site with 0.7 times PBS and 1%Penicillin streptomycin. Cover the top of the plate and screws with sterile bone wax to protect skin and muscles from irritation by the edges of the screws.
Place muscles and skin on top of the wax. Close the incision site with a size 7.0 synthetic suture using simple interrupted stitches. After surgery, to reawaken, place the animal in a fresh artificial-pond-water tank supplemented with penicillin, streptomycin, and butorphanol.
Micro CT and histological analysis facilitated the visualization of bone, fragment positions and gap sizes and the healing state. Notably, ambystoma Mexicanum showed no callous formation three weeks post-surgery contrasting with mammals. A cartilaginous callous was visible at three months and an ossified bony callus appeared at six months post-surgery.
