Induction of apoptotic cell death in rat thymus and spleen after a bolus injection of methamphetamine

We examined whether methamphetamine (MAP) induced apoptotic cell death in vivo. Male Wistar rats were injected intraperitoneally with 25 mg MAP/Kg body weight and were sacrificed at 4, 8 and 24 h. As early as 4 h after a single dose of MAP, DNA ladder bands representing DNA fragmentation into multiples of the internucleosomal DNA length of about 180 by were observed by gel electrophoresis in thymic and splenic DNA. DNA from control rats injected with 1 ml physiological saline/Kg body weight showed no ladder band patterns. The proportion of fragmented DNA from the thymus increased in a time-dependent manner up to 8 h and faint ladder band patterns were observed at 24 h, indicating that cell death via apoptosis occurred at an early stage and then apoptotic bodies were scavenged. DNA fragmentation in the thymus and spleen induced with MAP was also confirmed by the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) method in situ. In control thymus samples, stained cells were numerous in the cortex but sparse in the medulla. At the boundary area between the cortex and medulla, stained cells were seen as a layer. In the MAP-treated rats, stained cells were increased and dispersed equally in the cortex and medulla. In control spleen samples, stained cells were numerous in all areas excluding the germinal centers. Cells at the germinal centers were stained intensively in MAP-treated rat spleen. Light microscopical analyses allowed us to identify lymphocytes during the course of apoptotic cell death. Electron microscopic studies showed morphological landmarks for the process of cellular apoptosis in both organs e.g. lymphocytes with chromatin condensed into crescents at the periphery of the nuclei and apoptotic bodies. These results indicate that MAP induced cell death of the thymic and splenic lymphocytes via apoptosis.