AMPHIBIAN LIMB REGENERATION
213
and Salpeter suggest (1961, p. 288) that "it may represent an area of
accumulating secretion which is being discharged gradually through the
cytoplasmic membrane." Since wound epithelium intensely incorporates
methionine3 5
midine3
H (Hay and Fischman, 1961), this protein synthesis is apparently not involved with mitosis but may be associated with proteolytic
enzyme production.
Concomitant with the morphological changes in the limb stump during
demolition are changes in metabolism of the stump tissues. The act of
amputation not only removes distal limb structures but also interrupts
the continuity of the local circulatory system leading to local stasis of
blood flow adjacent to the wound. The resulting hypoxia of the tissues in
this region, along with anaerobic metabolism of muscle glycogen
(Schmidt, 1960), leads to the formation of large quantities of lactic acid
which tend to remain at the stump tip due to impairment of venous
return. It is also in this phase that Johnson and Singer (1964) detected
histochemically lactic dehydrogenase in the limb. Proteolytic enzyme
activity is high in this acidic environment. In an early study, Bromley
and Orechowitch (1934) found that proteolytic enzymes were 4 times
more active in remaining stump tissues after amputation than in these
tissues before amputation. The enzyme primarily involved is cathepsin.
As a result of its activities the intercellular materials of the tissues adjacent to the wound surface are histolyzed with a consequent doubling in
the amount of free amino acids in the stump. Also active at this time is
acid phosphatase which Schmidt and Weary (1963) believe is associated
with tissue and cellular autolysis (see Schmidt, 1966, for general review).
Quite recently, however, Weiss and Rosenbaum (1967) have attributed
the high acid phosphatase activity found in the wound epidermis of the
limb stumps of Ambystoma
larvae to phagocytic activity, since intracellular breakdown of injested particles is observed in the epithelial
cells. Nests of cells are also seen beneath the wound epidermis which
show intense acid phosphatase activity, "and such aggregates seem to
represent clusters of macrophages possibly engaged in various stages of
transport of materials gathered from points in the limb and shifted towards the apical epidermis" (Weiss and Rosenbaum, 1967, p. 211). Although the evidence is not conclusive, it does point to the possibility that
much of the debris resulting from trauma, and even dedifferentiation, is
transported by phagocytes and eliminated through the wound epithelium.
Thus a lysosomal mechanism is not necessarily implicated in limb regression. There is no reason to doubt, however, that cathepsinlike en-
213
and Salpeter suggest (1961, p. 288) that "it may represent an area of
accumulating secretion which is being discharged gradually through the
cytoplasmic membrane." Since wound epithelium intensely incorporates
methionine3 5
midine3
H (Hay and Fischman, 1961), this protein synthesis is apparently not involved with mitosis but may be associated with proteolytic
enzyme production.
Concomitant with the morphological changes in the limb stump during
demolition are changes in metabolism of the stump tissues. The act of
amputation not only removes distal limb structures but also interrupts
the continuity of the local circulatory system leading to local stasis of
blood flow adjacent to the wound. The resulting hypoxia of the tissues in
this region, along with anaerobic metabolism of muscle glycogen
(Schmidt, 1960), leads to the formation of large quantities of lactic acid
which tend to remain at the stump tip due to impairment of venous
return. It is also in this phase that Johnson and Singer (1964) detected
histochemically lactic dehydrogenase in the limb. Proteolytic enzyme
activity is high in this acidic environment. In an early study, Bromley
and Orechowitch (1934) found that proteolytic enzymes were 4 times
more active in remaining stump tissues after amputation than in these
tissues before amputation. The enzyme primarily involved is cathepsin.
As a result of its activities the intercellular materials of the tissues adjacent to the wound surface are histolyzed with a consequent doubling in
the amount of free amino acids in the stump. Also active at this time is
acid phosphatase which Schmidt and Weary (1963) believe is associated
with tissue and cellular autolysis (see Schmidt, 1966, for general review).
Quite recently, however, Weiss and Rosenbaum (1967) have attributed
the high acid phosphatase activity found in the wound epidermis of the
limb stumps of Ambystoma
larvae to phagocytic activity, since intracellular breakdown of injested particles is observed in the epithelial
cells. Nests of cells are also seen beneath the wound epidermis which
show intense acid phosphatase activity, "and such aggregates seem to
represent clusters of macrophages possibly engaged in various stages of
transport of materials gathered from points in the limb and shifted towards the apical epidermis" (Weiss and Rosenbaum, 1967, p. 211). Although the evidence is not conclusive, it does point to the possibility that
much of the debris resulting from trauma, and even dedifferentiation, is
transported by phagocytes and eliminated through the wound epithelium.
Thus a lysosomal mechanism is not necessarily implicated in limb regression. There is no reason to doubt, however, that cathepsinlike en-