The molecular pathways underlying the neuroprotective effects of preconditioning are promising, potentially drugable targets to promote cell survival. However, these pathways are complex and are not yet fully understood. In this study we have established a paradigm of hypoxic preconditioning based on a chick embryo model of normobaric acute hypoxia previously developed by our group. With this model, we analyzed the role of hypoxia-inducible factor-1α (HIF-1α) stabilization during preconditioning in HIF-1 signaling after the hypoxic injury and in the development of a neuroprotective effect against the insult. To this end, we used a pharmacological approach, based on the in vivo administration of positive (Fe(2+), ascorbate) and negative (CoCl(2)) modulators of the activity of HIF-prolyl hydroxylases (PHDs), the main regulators of HIF-1. We have found that preconditioning has a reinforcing effect on HIF-1 accumulation during the subsequent hypoxic injury. In addition, we have also demonstrated that HIF-1 induction during hypoxic preconditioning is necessary to obtain an enhancement in HIF-1 accumulation and to develop a tolerance against a subsequent hypoxic injury. We provide in vivo evidence that administration of Fe(2+) and ascorbate modulates HIF accumulation, suggesting that PHDs might be targets for neuroprotection in the CNS.
This paper describes modifications of the standard methods for obtaining a soluble nuclear fraction from embryonic brain tissue. The main improvements are: (1) the inclusion of a low speed centrifugation step to prevent the appearance of high density contaminants, (2) a sucrose density gradient to remove perinuclear mitochondria and ER membranes and (3) a protein extraction approach which significantly enhances protein yield. To demonstrate the effectiveness of the method, pellets were analyzed by light and electron microscopy and purity of the soluble extracts was immunologically tested. Finally, to illustrate the applicability of this approach, the induction of the transcription factor HIF-1 (hypoxia-inducible factor-1) was assessed by Western blot using soluble nuclear fractions and by immuno-electron microscopy using purified nuclear fractions, both obtained from the optic lobes of chick embryos. In conclusion, the procedure presently described appears to be reliable and convenient for obtaining a pure soluble nuclear fraction from a discrete amount of embryonic brain tissue.
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