N-tosyl-L-phenylalanyl-chloromethyl ketone eliminates the increase in caspase-3 and bcl-2 caused by brain injury in the newborn rat.

N-Tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) is neuroprotective in rat pups. We measured bcl-2, Bax and caspase-3 to determine the mechanisms. Seven-day-old rats had the right carotid artery ligated and were subjected to 2.5 h of 8% oxygen. Ten mg/kg of PTCK or vehicle was given intraperitoneally 15 min prior to hypoxia. At 24 h after hypoxia the brains were removed. Bcl-2 in the hippocampus increased from 0.149 +/- (SE) 0.023 in the shams to 0.289 +/- 0.037 with injury and vehicle (p < 0.05 vs. shams), which was reduced to 0.177 +/- 0.030 by TPCK ( p < 0.05 vs. vehicle). Bcl-2 in the cortex increased from 0.180 +/- 0.037 in the shams to 0.655 +/- 0.078 with injury and vehicle (p < 0.01 vs. shams), which was reduced to 0.354 +/- 0.035 by TPCK (p < 0.01 vs. vehicle). Bax, measured only in the mitochondrial enriched fraction of the cortex, was unchanged. Caspase-3 activity increased with injury to 245 +/- 38% of baseline in the hippocampus (p < 0.01) and to 261 +/- 69% in the cortex (p < 0.01). Treatment with TPCK reduced this to 132 +/- 16% in the hippocampus (p < 0.01 vs. vehicle) and 140 +/- 14% in the cortex (p < 0.05 vs. vehicle). In this experiment TPCK reduces bcl-2 and caspase-3 concentration in animals who have been shown in our previous studies to be protected by TPCK from hypoxic ischemic brain injury. This is consistent with the hypothesis that TPCK produces neuroprotection by blocking the apoptotic cascade between bcl-2 and caspase-3.