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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
NOTE: Donor rats are allowed free access to food and water prior to skin and muscle donation procedures. Euthanasia is performed under deep anesthesia followed by intracardiac potassium chloride injection with a secondary method of bilateral pneumothorax. Any strain of rat can theoretically be utilized with this experiment; however, our laboratory has achieved consistent results in both male and female Fischer F344 rats (~200-250 g) at two to four months of age. Donor rats must be isogenic to the experimental rats.
1. Preparation of the dermal graft
- Anesthetize the donor rat in an induction chamber utilizing a solution of 5% isoflurane in oxygen at 0.8-1 L/min. Once the rat has been anesthetized, remove from induction chamber and place on a rebreathing nose cone, lowering the isoflurane to 2-2.5% for maintenance of anesthesia.
- Administer a solution of 0.02-0.03 mL Carprofen (50 mg/mL) in 0.2 mL of sterile saline subcutaneously between the shoulder blades for analgesia.
- Apply artificial tears ointment to both eyes to prevent corneal ulcers.
- Using clippers, shave the entire lower hindlimb(s), ankle region, and sides of paw(s).
- Cleanse chosen hindlimb and plantar surface of paw with alcohol, followed by iodopovidone solution, ending with a final cleanse with alcohol to remove residual iodopovidone.
- Using a hand-held micro motor high-speed drill with a removable round fine grit polishing stone (4000 rpm), burr the plantar surface of the paw to remove the epidermis. While burring, apply drips of saline so as not to burn the skin. The underlying dermis will have a shiny appearance with pinpoint bleeding.
- Apply a tourniquet to the lower extremity to slow blood flow.
- Remove the plantar skin sharply with a #15 scalpel and place it in saline-moistened gauze to prevent desiccation. Some tendinous and connective tissue will inherently be removed with the skin in this step and will be removed later.
- Apply a gauze wrap to the bleeding foot to slow the hemorrhage. Repeat steps 1.5-1.9 if doing two constructs.
- Under a microscope (20x magnification), remove the tendinous and connective tissue from the deep layer of the skin graft using microscissors. Take care not to make holes in the graft. The thinned dermal graft should be slightly opaque containing only dermis, measuring approximately 0.5 cm x 1.0cm in size.
- Place in saline-moistened gauze until ready for C-RPNI construct fabrication. Grafts should be utilized within 2 hours of harvest.
2. Preparation of the muscle graft
- Make a longitudinal incision along the anterior aspect of the lower hindlimb from just above the ankle to just below the knee with a #15 scalpel. Dissect through the subcutaneous tissue to expose the underlying musculature.
- At the distal aspect of the incision, expose the tendinous insertions of the lower limb musculature. Tibialis anterior (TA) is typically the largest and most anterior of the muscles, and just underneath and posterior to this muscle lies the extensor digitorum longus (EDL). Isolate the distal EDL tendon from the other tendons in the area, taking care not to incise its insertion at this point.
- Ensure isolation of the correct tendon by inserting both tines of a forceps underneath the tendon and exerting upward pressure by opening the forceps to cause tendon excursion. Manipulation of this tendon should cause all of the toes to extend simultaneously.
- Perform a distal tenotomy with sharp iris scissors and separate the muscle from the surrounding tissues bluntly with tenotomies (or other blunt-tipped scissors), working proximally to find the tendinous origin.
- Once the proximal tendon is visualized, again perform a tenotomy utilizing sharp iris scissors. Place the muscle graft in a saline-moistened gauze to prevent desiccation.
- Once all desired grafts have been removed from a donor rat, euthanize primarily by intracardiac KCl injection (1-2 mEq K+/kg) followed by secondary euthanasia with bilateral puncture pneumothorax with a #15 blade.
3. Common peroneal nerve isolation and preparation
- Anesthetize and provide analgesia to the experimental rat according to the protocol outlined in steps 1.1-1.3.
- Shave the desired thigh and cleanse with alcohol, betadine, and end with alcohol to remove traces of betadine.
- Move the animal from the surgical prep table to the surgical microscope table and place it on a heating pad with a temperature probe for body temperature maintenance. Maintain isoflurane at 2-2.5% and oxygen at 0.8-1 L/min.
- Mark the incision, extending from just distal to the sciatic notch to the inferior portion of the knee. This marking should be inferior to, and angled away from, the femur. Make the incision with a #15 blade, incising through the underlying biceps femoris fascia.
- Carefully dissect through the biceps femoris muscle with either a hemostat or blunt-tipped microscissors to the space underlying the biceps femoris.
NOTE: The sciatic nerve travels approximately in the same direction as the initial incision that was made. There are three branches, typically with the sural nerve posterior and the common peroneal and tibial nerve traveling superficial and deep to the knee, respectively. - Following identification of the common peroneal (CP) nerve, using a pair of micro-, fine-tipped forceps and micro-scissors, carefully isolate the CP nerve from the other sciatic branches and remove any lingering connective tissue distally.
- At the point where the nerve crosses the surface of the knee, sharply transect the nerve with a pair of microscissors.
NOTE: Using sharp scissors is extremely important in this step, as causing significant trauma to the nerve could increase the risk of neuroma formation. - Carefully free any remaining connective tissue from the CP nerve and work proximally to free the nerve to a length of approximately 2 cm.
4. C-RPNI construct fabrication
- Remove the muscle graft from saline-moistened gauze and remove all central tendinous tissue as well as a small central segment of epimysium. Leave the tendinous ends intact.
- Using an 8-0 nylon suture, secure the epineurium of the transected end of the CP nerve to the area of the muscle graft devoid of epimysium with two interrupted stitches on either side of the nerve.
- Secure the muscle graft to the femur periosteum with a single 6-0 nylon interrupted stitch both proximally and distally, with the nerve-muscle junction facing away from the femur.
NOTE: Secure the muscle so that it is at normal, relaxed length. Try not to stretch the muscle significantly or leave too much laxity when securing. - Place an 8-0 nylon stitch at the inferior, central margin of the muscle graft epimysium, securing it to the CP nerve epineurium in a way as to create laxity in the nerve within the muscle graft and help to relieve any future tension it may be exposed to with later ambulation.
- Remove the skin graft from the saline-moistened gauze and arrange it on the muscle graft in such a way to completely cover the nerve and the majority of the muscle. Ensure that the deep margin of the dermis is resting on the muscle. Trim any dermis that extends beyond the border of the muscle.
- Secure the skin graft to the muscle graft circumferentially using 8-0 nylon interrupted sutures. Typically, 4-8 total sutures are used depending on the size of the construct.
- Close the biceps femoris fascia over the construct in a running fashion with 5-0 chromic suture.
- Close the overlying skin with a 4-0 chromic suture in running fashion.
- Swab the surgical area with an alcohol pad and apply antibiotic ointment.
- Cease inhalational anesthetic and allow the rat to recover with food and water sources separate from cage mates.