Histochemical approaches to characterize apoptotic cells.

Apoptosis is a morphologically distinct set of cell death process, discriminated from necrosis, and is induced through the activation of intrinsic cell-suicide program. To understand better the fundamental process of apoptosis, which is expected to promote us to manipulate cell death to our advantages, cellular analysis of cell death is required since apoptotic cells often co-exist with necrotic ones in tissues. In this study, we attempted several histochemical trials to define a difference between apoptosis and necrosis using hydrocortisone-treated rat thymus and CCl4-treated rat liver as models of apoptosis and necrosis, respectively. When the fresh frozen sections were stained for acid phosphatase activity, the staining in necrotic hepatocytes was lost, while that in apoptotic thymocytes was well retained, reflecting the integrity of lysosome in apoptosis. In situ nick translation (ISNT) as well as TUNEL, which detect DNA strand breaks, also allowed us to differentiate apoptotic cells from necrotic cells in the frozen sections; the apoptotic nuclei were stained only when the sections were treated with proteinase K (PK), whereas the necrotic nuclei were stained without PK treatment. However, in paraffin sections apoptotic nuclei were stained by these methods without PK treatment. Therefore, the criterion to discriminate both types of cell death did not hold in this case. We also addressed whether the expression of Fas or Fas ligand is a useful histochemical marker of apoptosis. Consequently, a positive correlation was confirmed in ischemia-reperfusion mouse kidney and in ulcerative colites, while it could not be always generalized. Thus, in fact any one of these methods is not absolute singly, but rather mutually supplementary. Currently, for a precise description of cell death, it seems essential to combine various methods including morphological and biochemical approaches.