Tetrazolium Salt and Electron-Microscopic Studies of Cellular Degeneration and Necrosis in the Interdigital Areas of the Developing Chick Limb

Abstract

Cellular degeneration and necrosis were studied in the interdigital areas of developing hind limb buds of normal chick embryos by means of enzyme-specific tetrazolium salts and electron microscopy. Using succinic acid as a substrate and the tetrazolium salt, nitro blue tetrazolium, succinate dehydrogenase was specifically demonstrated via a colour reaction in which degenerating and necrotic cells--those with no enzyme activity--did not stain, while those with enzyme activity stained deeply blue-black. The interdigital cells exhibited near absent levels of succinate dehydrogenase as early as stage 26-27, one to two days prior to when morphological evidence of degeneration and necrosis was present. It was postulated that the mechanism of cell death resulted from decreased activity and/or loss of strategic cellular enzymes such as succinate dehydrogenase, with a subsequent fall in the cellular adenosine triphosphate (ATP) level and a resultant compromise in vital cellular processes, eventually leading to cell death. The evidence indicated that ‘biochemical degeneration’ occurred prior to morphological changes in cells. The ultrastructural events of degenerating and necrotic cells were also studied. Most ‘dying’ cells observed had already been phagocytosed and were observed in various stages of degeneration. The degenerating cells exhibited both nuclear and cytoplasmic changes. There was evidence of active intracellular digestion within the phagocytes. Numerous lysosomes were observed within these cells, and some appeared fused with the digestive vacuole's membranes. The origin of the phagocytes was not determined. No definite information was obtained concerning the utility of cellular degeneration and necrosis in the interdigital areas of the hind limb buds. It appeared to be at least partially causal in separation of the digits.