Abstract:OBJECTIVE: Activation of calcium-dependent neutral proteinase, Caplain, can induce apoptosis of neuron and Caplain inhibitor-3 (MDL28170) has protective effects against brain ischemia in adult animals. Whether it also has protective effects on neonatal animals with hypoxic-ischemic brain damage (HIBD) is has not been determined. In this study, the effect of MDL28170 on Caspase-3 expression and apoptosis in hippocampal CA1 region of neonatal rats after HIBD was examined to explore its neural protective effect on neonatal animals and the possible underlying mechanism. METHODS: Seven-day-old Sprague-Dawley rat pups were randomly assigned into three groups: HIBD (n=40), MDL (n=40) and Control (n=8). The pups in the first two groups were subjected to unilateral ligation of the right carotid artery followed by 2 hrs of hypoxia (8% O_2). The pups in the MDL group were intraperitoneally injected with MDL28170 (50-mg/kg) at 0, 2 and 4 hrs after hypoxia-ischemia(HI), while those in the Control and HIBD groups were intraperitoneally injected with normal saline instead. The rats in the HIBD and MDL groups were sacrificed at 6, 12, 24, 48 and 72 hrs after HI (8 rats in each group at each time point). Immunohistochemistry and terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling staining (TUNEL) were used to detect the Caspase-3 expression and neuronal apoptosis in hippocampal CA1 region. RESULTS: In the HIBD group, Caspase-3 expression in hippocampal CA1 region increased at 6 hrs after HI compared with the Control group (13.4±3.5/HP vs 2.6±0.6/HP,P=0.028), peaking at 48 hrs (27.1±4.1/HP) and remaining higher at 72 hrs (22.6±4.8/HP,P<0.001). The number of TUNEL-positive neurons in the HIBD group increased at 6 hrs after HI, and were significantly greater than those of the Control group at 12 hrs (25.0± 1.7/HP vs 2.3±1.5, P<0.001). At 48 hrs after HI, the number of TUNEL-positive neurons peaked (67.8±2.6/HP) and remained greater at 72 hrs (44.3±6.8/HP). The treatment with MDL28170 attenuated the Caspase-3 expression and reduced the number of TUNEL-positive neurons within 48 hrs after HI when compared with the HIBD group (P<0.05), but the effect significantly decreased at 72 hrs. CONCLUSIONS: MDL28170 can inhibit the Caspase-3 expression and reduce the apoptotic cells in hippocampal CA1 region, thereby yielding protective effects against HIBD in neonatal rats.
