Introduction
Preconditioning with hypoxia can protect the brain against a subsequent hypoxic-ischaemic insult. Hypoxia alone is not sufficient to cause neuronal injury, however can induce changes in gene expression and intracellular signalling pathways. The expression of the hypoxia-inducible transcription factor (HIF-1) 1 and several HIF-1 target genes 2 are up-regulated after hypoxic preconditioning in neonatal rat brain. Recent evidence has demonstrated that a family of HIF prolyl hydroxylase enzymes (Egln1,2 &3) are important in regulating HIF-1 function. Under normoxic conditions, Hif-1α protein is hydroxylated, allowing binding of von Hippel Lindau protein, making Hif-1α a target for proteosomal degradation. Enzymatic activity of prolyl hydroxylases is oxygen-dependent, therefore under hypoxic conditions, degradation of Hif-1α is prevented, allowing accumulation of Hif-1α protein, dimerization with Hif-1β and the HIF-1 complex can bind to the hypoxia-responsive element of various HIF-1 target genes (VEGF, GLUT-1, EPO) 3 . We have investigated whether hypoxic preconditioning can affect levels of Egln1 protein in neonatal rat brains.
Methods
Sprague Dawley rat pups (postnatal day 6) were exposed to preconditioning with hypoxia (3 h, 8% oxygen) or normoxia (3 h, room air). On postnatal day 7, pups were subjected to occlusion of the left common carotid artery, followed by a 2.5 h hypoxic exposure. At 5 days after hypoxic-ischemic insult, pups were sacrificed and brains removed to examine neuroprotective effect of hypoxic preconditioning. A further group of pups were exposed to hypoxia preconditioning (p6) and at various times (0, 0.5 h, 2, 4, 16 and 24 h) after cortical tissue was obtained for Western blot analysis of Egln1 protein levels.
Results
Preconditioning with hypoxia afforded significant protective effect against hypoxic-ischemic insult in neonatal rat brain. Following exposure to hypoxia preconditioning, there was an increase in Egln1 protein compared with normoxic control tissue (80–120% increase, sustained from 0.5 h–24 h after reoxygenation; p<0.05, ANOVA).
Conclusions
Modulation of HIF-1 function by prolyl hydroxylation is likely to be involved in the protective effect conferred by hypoxic preconditioning in neonatal rat brain. We have observed that Egln1 protein expression remains elevated up until 24 hours after hypoxic preconditioning – which could possibly be regulated by HIF-1 at the transcriptional level.