Dimitrios Angelis, Tania D. Fontánez-Nieves, Maria Delivoria-Papadopoulos

Hypoxia induces a cerebral inflammatory response, which contributes to brain injury. Inflammasomes are complex intracellular molecular structures that initiate the inflammatory cascade. Caspase-1 and interleukin 1-β (IL-1β), have been established as markers of inflammasome activation. Src kinase, a cytosolic non-receptor protein tyrosine kinase, is linked to cell proliferation and differentiation and is up regulated during hypoxia. The role of Src kinase in the above pathway is not fully understood. The present study tests the hypothesis that inhibition of Src kinase, by a selective inhibitor, PP2, will prevent the activation of caspase-1 and production of IL-1β acutely, as well as at 1 and 15 days after hypoxia in the cerebral cortex of the newborn piglet. Piglets were divided into: Normoxia (Nx), Hypoxia acute (Hx), Hypoxia-day 1 (Hx-day 1), and Hypoxia day 15 (Hx-day 15). Piglets pretreated with Src kinase inhibitor, PP2, 1 mg/kg IV, 30 min prior to hypoxia were divided into: Hypoxia acute (Hx + PP2), 1 day (Hx + PP2-day 1), and day 15 (Hx + PP2-day 15). Hypoxia was induced by exposing the piglets to an FiO2 of 0.07 for 1 hour. Caspase-1 activity and expression were determined with spectrophotometry and Western blot respectively, while IL-1β levels were measured by solid phase ELISA. Caspase-1 activation was achieved immediately (within 1 h) after hypoxia and persisted for 15 days. IL-1β level was also increased after hypoxia reaching a maximum level at 24 h following hypoxia and returned to baseline by 15 days. Administration of PP2 attenuated the activity acutely, but not the expression of the caspase-1. IL-1β level at 24 h after hypoxia returned to baseline in piglets that were pretreated with PP2. We provide evidence that inhibition of Src kinase in the acute phase after hypoxia involves changes in the production or processing of caspase-1 subunits. Our data suggest that Src kinase mediates hypoxia-induced caspase-1 activation in the cerebral cortex of newborn piglets. Inhibition of Src kinase may attenuate the neuroinflammatory response and could represent a potential target for neuroprotection after hypoxic injury.