Yusuke Iwata1, Olivier Nicole2, David Zurakowski1, Richard A. Jonas1; 1Children's National Heart Institute, Washington, DC; 2UMR-CNRS 6185, Université Caen, Caen, France
Objective: Aprotinin is widely used in cardiac surgery for its anti-inflammatory and hemostatic benefits. Recent reports in the neuroscience literature describe neuroprotective effects of other serine protease inhibitors via reduced excitotoxic cell death, a common pathway causing cytotoxic edema induced in various neuropathologic conditions. The purpose of this study was to investigate whether Aprotinin has direct neuroprotective effects against glutamatergic excitotoxicity using both mixed and near-pure neuronal cell culture models.Methods: Mixed cortical cultures containing neuronal and glial cells were prepared from fetal mice at 13-15d gestation, plating on a layer of confluent astrocytes from 1-3d postnatal pups. Near-pure neuronal culture containing less than 5% astrocytes were obtained from same gestational stage, plating in multiwell vessels previously coated with poly-D-lysine and laminin. Both cultures were used at 12-14 days in vitro. Slow triggered excitotoxicity was induced at 37°C by 24 hour exposure to 12.5µM N-methyl-D-aspartate (NMDA) or 50µM kainate. Neuronal death was quantified by measuring the release of lactate dehydrogenase from damaged cells into the bathing medium. Data were analyzed using ANOVA with Bonferroni post-hoc comparisons.
Results: Aprotinin at a clinically relevant concentration of 100 KIU/ml significantly protected neurons against NMDA-induced neuronal death in both pure and mixed cultures (P < .001, Fig 1). Aprotinin also reduced neuronal cell death induced by kainate from 36% to 23% in mixed cortical culture (P = .008) and from 40% to 27% in near-pure culture (P = .015), indicating that the neuroprotective effects of aprotinin are mediated directly through neurons.
Conclusion: Aprotinin provides direct neuroprotection against glutamatergic excitotoxicity as demonstrated by reduced neuronal cell death in near-pure neuronal cell culture. Additional studies are needed to evaluate the potential of aprotinin to reduce neurological injury in patients at high risk of cerebral injury, including those undergoing circulatory arrest.
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