Microscopic observations show invasion of inflammatory cells in the limb blastema and epidermis in pre-metamorphic frog tadpoles which destroy the Apical Epidermal CAP and impede regeneration.

Some limb regeneration in tadpoles of Rana dalmatina occurs at stages 44-48 when small hind-limbs are present while scarring occurs at stages 51-52 when forelimbs have developed and metamorphosis is approaching. Ultrastructural analysis of cells forming the regenerating blastema detects mesenchymal cells and an Apical Epidermal Cap (AEC) in regenerating limb blastema 5-6 days post-amputation at stages 46-48. In contrast, granulocytes and numerous macrophages and lymphocytes prevail over mesenchymal cells in limb blastema at stages 51-52, which are destined to form scars. An increase in inflammatory cells in limb blastema prior to metamorphosis suggests a negative influence of immune cells on limb regeneration. Inflammatory cells invade the apical wound epidermis where stem keratinocytes are likely destroyed, impeding the formation of an AEC, the microregion which leads to limb regeneration. The invasion of immune cells, however, may also represent a physiological consequence of the death of cell populations in the tadpoles occurring with approaching metamorphosis. The passage from an aquatic to a terrestrial life in this frog elicits the typical amniote scarring reaction after wounding, and the limb cannot regenerate. The present observations sustain the hypothesis that the evolution of the adaptive immunity in tetrapods while efficiently preserving adult self-condition, determined the loss of tissue regeneration since the embryonic antigens evocated in blastema cells are removed by immune cells of the adult.