Limb Regeneration AMPUTATION Anaesthetize axolotl with MS222 or
Limb Regeneration AMPUTATION Anaesthetize axolotl with MS-222 or benzocaine in water until its “righting reflex” disappears. Place the animal on a short stack of clean, wet paper towels. Amputations are made with a clean scalpel. Stretch the target limb out and press down firmly and quickly with scalpel in the desired location on the limb. Very little, if any, bleeding should occur due to a very rapid vasoconstriction reflex. Usually only a drop or two of blood is lost. Bubbling or cooling the water during recovery will help the axolotl revive from the anesthesia.
Limb Regeneration MIGRATION Within an hour post-amputation, basal epidermal cells next to the wound site migrate to the wound to cover it with a single-cell layer of epidermal cells. This step is somewhat similar to the clot-formation reaction in mammals, but in urodeles, a clot does not form.
Limb Regeneration WOUND EPITHLIUM Once the tip of the “stump” is covered over with epithelial cells, this cell layer is called the wound epithelium.
Limb Regeneration IMMUNE RESPONSE Once the wound epithelium is completed, there is a minor immune response, which resolves quickly. During the immune response, Macrophages are released. These macrophages release enzymes (collagenases, etc. ) that degrade the extra cellular matrix of the cells near the amputation site, reverting the extra cellular matrix to its embryonic state.
Limb Regeneration DEDIFFERENTIATION As the extra cellular matrix is degraded, the freed cells dedifferentiate into mesenchyme. The mesenchymal cells directly touch the wound epithelium -there is no dermis present at this point.
Limb Regeneration CELL PROLIFERATION Direct contact between the mesenchymal cells and the overlying wound epidermis keeps the proliferating mesenchymal cells from re-differentiating. The mesenchymal cells enter the cell cycle and proliferate in great numbers, forming a blastema.
Limb Regeneration REDIFFERENTIATION Re-differentiation occurs from the most proximal site outward towards the distal edge of the limb. The differentiated cells “know” where on the limb they are, so that when they re-differentiate, they become the appropriate cells for their position on the limb. Retinoic Acid interferes with this locational memory.
Limb Regeneration LIMB OUTGROWTH Once the cells are re-differentiated, the limb grows just as it would normally.
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