Sara Fiszer de Plazas

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 (Fe²⁺, ascorbate) and negative (CoCl₂) 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 Fe²⁺ and ascorbate modulates HIF accumulation, suggesting that PHDs might be targets for neuroprotection in the CNS.

It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1–NR2A and NR1–NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis.

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.

The neuroprotective effects of oestrogens have been demonstrated against a variety of insults, including excitotoxicity, oxidative stress and cerebral ischemia under certain conditions. However, the molecular mechanisms underlying oestrogen neuroprotection are still unclear. We aimed to determine whether 17β‐oestradiol (E₂) administration post‐hypoxia (p‐hx) was neuroprotective and whether these actions were mediated through oestrogen receptors (ER). For this purpose, 12‐embyonic day‐old chickens were subjected to acute hypoxia [8% (O₂), 60 min], followed by different reoxygenation periods. To test the neuroprotective effect of E₂ and its mechanism, embryos were injected 30 min after the end of hypoxia with E₂ alone or with ICI 182 780, a competitive antagonist of ER. Cytochrome c (cyt c) release, an indicator of mitochondrial apoptotic pathway, was measured by western blot in optic lobe cytosolic extracts. DNA fragmentation by TUNEL fluorescence and caspase‐3 fragmentation by immunofluorescence were detected on optic lobe sections. Acute hypoxia produces a significant increase in cyt c release from mitochondria at 4 h p‐hx, followed by an increase in TUNEL positive cells 2 h later (6 h p‐hx). Administration of E₂ (0.5 mg/egg) produced a significant decrease in cytosolic cyt c levels at 4 h p‐hx, in casapse‐3 activation and in TUNEL positive cells at 6 h p‐hx compared to vehicle treated embryos. In the E₂‐ICI 182 780 treated embryos, cyt c release, caspase‐3 fragmentation and TUNEL positive cells were similar to the hypoxic embryos, thus suggesting the requirement of an E₂–ER interaction for E₂ mediated neuroprotective effects. In conclusion, E₂ prevents hypoxia‐induced cyt c release and posterior cell death and these effects are mediated by oestrogen receptors.

NO-mediated toxicity contributes to neuronal damage after hypoxia; however, the molecular mechanisms involved are still a matter of controversy. Since mitochondria play a key role in signalling neuronal death, we aimed to determine the role of nitrative stress in hypoxia-induced mitochondrial damage. Therefore, we analysed the biochemical and ultrastructural impairment of these organelles in the optic lobe of chick embryos after in vivo hypoxia-reoxygenation. Also, we studied the NO-dependence of damage and examined modulation of mitochondrial nitric oxide synthase (mtNOS) after the hypoxic event. A transient but substantial increase in mtNOS content and activity was observed at 0-2 h posthypoxia, resulting in accumulation of nitrated mitochondrial proteins measured by immunoblotting. However, no variations in nNOS content were observed in the homogenates, suggesting an increased translocation to mitochondria and not a general de novo synthesis. In parallel with mtNOS kinetics, mitochondria exhibited prolonged inhibition of maximal complex I activity and ultrastructural phenotypes associated with swelling, namely, fading of cristae, intracristal dilations and membrane disruption. Administration of the selective nNOS inhibitor 7-nitroindazole 20 min before hypoxia prevented complex I inhibition and most ultrastructural damage. In conclusion, we show here for the first time that hypoxia induces NO-dependent complex I inhibition and ultrastructural damage by increasing mitochondrial NO in the developing brain.

Exposure of the CNS to hypoxia is associated with cell death. Our aim was to establish a temporal correlation between cellular and molecular alterations induced by an acute hypoxia evaluated at different post-hypoxia (p-h) times and at two stages of chick optic lobe development: embryonic days (ED) 12 and 18. TUNEL assays at ED12 disclosed a significant increase (300%) in pyknotic cells at 6 h p-h, while at ED18 no morphological changes were observed in hypoxic versus controls. At ED12 there was a significant increase (48%) in Bcl-2 levels at the end of the hypoxic treatment, followed by a significant increase of active caspase-9 (49%) and active caspase-3 (58%) at 30 and 60 min p-h, respectively, while at ED18 no significant changes were observed. These findings indicate that prenatal hypoxia produces an equilibrated imbalance in both pro- and anti-apoptotic proteins that culminates in a process of cell death, present at earlier stages of development.

It has been demonstrated that the CNS is severely affected by hypoxic-ischemic insults during the prenatal-perinatal period, including imbalance in excitatory and inhibitory neurotransmitter release....