Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, U.S.A.

Correspondence: John M Hallenbeck, Stroke Branch National Institute of Neurological Disorders and Stroke, National Institutes of Health, 36 Convent Drive MSC 4128, Building 36/Room 4A03, Bethesda, MD 20892-4128, U.S.A.

Received 4 August 2000; Revised 26 September 2000; Accepted 3 October 2000.

Abstract

Preconditioning brain with tumor necrosis factor alpha (TNF-) can induce tolerance to experimental hypoxia and stroke and ceramide is a downstream messenger in the TNF- signaling pathway. A hypoxic–ischemic (HI) insult in the immature rat injures brain primarily through apoptosis. Apoptosis is regulated by Bcl-2 family proteins. The authors explored whether ceramide protects against HI in the immature rat, and whether Bcl-2 family protein expression is involved. Hypoxia–ischemia was produced in seven-day-old rats by ligating the right carotid artery, followed by 2 hours of 8% oxygen exposure. Thirty minutes after HI, C₂-ceramide (150 g/kg) was injected intraventricularly. Infarct volume was measured 5 days later. C₂-ceramide reduced HI-induced brain damage by 45% to 65% compared with HI/dimethyl sulfoxide (DMSO) (vehicle control) or HI only groups. In separate experiments, brains of sham-operated control and HI only animals and animals subjected to HI plus C₂-ceramide or DMSO infusion were sampled 6 hours, 24 hours, and 5 days after treatments and analyzed for Bcl-2, Bcl-xl, and Bax expression (Western blotting), and apoptosis (TUNEL assay). Augmented Bcl-2 and Bcl-xl levels in the C₂-ceramide treated group were associated with a significant decrease in TUNEL-positive cells. The results support a protective role for ceramide in neonatal HI.