APOPTOTIC, INJURY-INDUCED CELL-DEATH IN CULTURED MOUSE MURINE MOTOR-NEURONS

In order to develop in vitro models of CNS injury, astrocytes have bee n mechanically injured in culture to study reactive astrocytosis. Howe ver, scratch injury models of pure neuronal cultures have not yet been exploited to study programmed cell death (PCD). For this study, we ex amined model motor neurons (NSC19 cells) in culture and found time-dep endent cell death in proximity (within 2.5 mm) to a physical scratch i njury. Injury-induced cell death was apoptotic verified by positively- stained nuclei using both the in situ end-labeling (ISEL) procedure an d Hoechst 33342. Unexpectedly, cells proximal to the injury site were not affected by the injury until 3 days later suggesting that adjacent motor neuron loss was dependent on a 'death signal' produced by direc t injury to sister neurons. 'Executioners' in apoptosis include free r adicals, cell cycle kinases and cysteine proteases (caspases). Extrace llular serine proteases, such as thrombin and granzyme B, may activate such intracellular pathways and several inhibitors (serpins), such as CrmA, are effective in blocking apoptosis. Since protease nexin I (PN I), a serpin homologous with CrmA, prevents apoptosis of lumbar motor neurons and is increased after nerve injury, we examined mRNA by RT-PC R for PNI expression. Of interest, although we were unable to find sig nificant levels of PM message in NSC19 cells, we did detect it in the parent neuroblastoma. (C) 1997 Elsevier Science Ireland Ltd.