N-tosyl- l-phenylalanyl-chloromethyl ketone reduces ceramide during hypoxic–ischemic brain injury in newborn rat

Abstract

N-tosyl- l-phenylalanyl-chloromethyl ketone (TPCK) suppresses apoptosis and protects neurons from damage in animal models. TPCK is thought to act by inhibiting ceramide production by sphingomyelinase. Ceramide is a proapoptotic intracellular signal that is involved in the cerebral ischemia. We wished to see whether ceramide contributes to TPCK's neuroprotective effects in vivo. Seven-day-old rat pups had the right carotid arteries permanently ligated followed by 2.5 h of hypoxia (8% oxygen). TPCK (10 mg/kg, n = 62) or vehicle ( n = 63) was administered by i.p. 5 min prior to hypoxia. The level of ceramide in brain cortex both in lesioned and unlesioned hemispheres was measured at 8 h, 18 h, 24 h, 2 and 5 days after hypoxia–ischemia using reversed phase high performance liquid chromatography. The level of ceramide significantly increased due to hypoxic–ischemia at 18, 24 h and 2 days after hypoxia ( P < 0.05 or P < 0.01) but not at 8 h or 5 days after hypoxia as compared to the contralateral hemisphere or a sham group. Pretreatment with TPCK reduced this increase. We also examined the level of sphingomyelin and the activities of the ceramide synthesizing sphingomyelinase enzymes by thin layer chromatography. The activities of acidic and neutral sphingomyelinase significantly increased due to hypoxic ischemia at 24 h after hypoxia. TPCK significantly reduced this increase ( P < 0.05 vs. vehicle) but did not affect the level of sphingomyelin. The results are consistent with the hypothesis that ceramide is involved in TPCK's neuroprotective effects in hypoxic–ischemic brain injury in the newborn rat.