Axolotl limb regeneration requires a specialized wound epidermis. This surgery inhibits wound epidermis formation and can be paired with molecular and functional techniques to study limb regeneration. The main advantage of this protocol is that it is efficient and technically reproducible.
This surgical method provides researchers with a tool to interrogate wound epidermis function and advance our understanding of why this structure is critical for regeneration. After ensuring that the axolotl is fully anesthetized, perform the limb amputation at the distal end of the zeugopodial skeletal elements using the dissecting scissors. Then, use spring scissors to make a small incision on the ventral portion of the skin.
Using the forceps, carefully peel back the skin to approximately the midline of the zeugopodial skeletal elements, exposing the underlying limb tissues while making sure to not damage the skin. If possible, use a duller pair of forceps when handling the full-thickness skin flap to prevent damage. Amputate the exposed underlying limb tissues at the midline of the zeugopod with the surgical scissors, then push back the muscle tissue with the surgical scissors and trim the exposed bone.
Carefully pull the extra full-thickness skin over the amputation plane to cover the exposed underlying tissues, making sure that the skin flap maintains its structural integrity. Suture it to the ventral full-thickness skin. Using the forceps and curved spring scissors, suture the remaining right and left sides of the skin flap into the underlying ventral portions of intact skin in a crisscross manner.
When finished, make sure that no exposed underlying tissues can be seen, and that the sutures are tied tight and knotted at least three times. As an optional internal control, perform an amputation on the contralateral limb by amputating it at mid-zeugopod level with surgical scissors, then push back the muscle tissue with surgical scissors and trim the exposed bone. Place the delicate-task wipe at the bottom of a container with sulfamerazine solution on ice.
Then, place the axolotl into the container and gently wrap the exposed ends of the wipe around the top of the animal to keep it well hydrated. Leave it on ice for 30 minutes to one hour to ensure minimal movement during recovery from anesthesia, then place it into a static housing container with 0.5%sulfamerazine solution. Axolotls must remain in this solution for the first 24 hours after surgery in order to prevent infection.
This surgical protocol allows for the complete inhibition of wound epidermis formation, and ultimately, limb regeneration. It is important to inspect the sutured limb daily. If one or more of the sutures pop out, a wound epidermis can form, resulting in either a small or large blastema and an unsuccessful surgery.
Sectioning sagittally through the sutured limb tissue and performing histological analyses at any time point can also verify the presence of the dermis from the full skin flap and circling the entire amputation plane in the absence of a wound epidermis. When attempting this procedure, it is important to make sure that the skin flap maintains its structural integrity and covers the amputation plane. Knotting the sutures tightly and making sure the flap is secure is also critical to the success of the protocol.
This method can be paired with downstream analyses, including RNA sequencing or functional techniques including ectopic genetic over-expression, to identify novel molecular regulators of wound epidermis function and limb regeneration.
