Electrical Stimulation for Limb Regeneration

Electrical stimulation was applied using a purpose-built bimetallic device and surgeries were performed as described previously^{32 (link)}. Animals were euthanized (CO₂ inhalation) at 3-, 7- and 28- days post amputation, weighed, and the limb stumps were collected and carefully examined macro- and microscopically. In the macroscopic examination, the skin covering the limb stumps of the EStim and sham-treated animals was carefully incised and the EStim device and corresponding electrodes were controlled for integrity and carefully removed. In addition, all limbs were inspected for signs of infection and/or tumors. The specimens were fixed in Zinc-Formal-Fixx, (Thermo Electron, USA) for 24hrs and stored for subsequent histomorphometric and immunohistological analysis or stored in liquid nitrogen for subsequent RNA isolation.