Elsevier

Brain Research

Volume 1199, 14 March 2008, Pages 27-36
Brain Research

Research Report
HIF-1-regulated vasoactive systems are differentially involved in acute hypoxic stress responses of the developing brain of newborn mice and are not affected by levetiracetam

https://doi.org/10.1016/j.brainres.2007.12.069Get rights and content

Abstract

Hypoxia-inducible transcription factor-1 (HIF-1) is critically involved in adaptive endogenous mechanisms to hypoxic brain injury by transcriptional activation of specific target genes that restore oxygen supply. Exogenously, neuroprotective properties of levetiracetam (LEV) have been suggested in experimental cerebral ischemia and epilepsy. We aimed to elucidate 1) effects of acute hypoxic distress on HIF-1 and vasoactive target genes, and 2) effects of LEV on HIF-1-regulated mechanisms in the brain at early developmental stages. To this end, we studied the impact of hypoxia in the presence or absence of LEV on the O2-dependent HIF-1α subunit as well as on VEGF and iNOS in the developing brain of normoxic and hypoxic mice. C57BL/6 mice (P0, P7) were treated with saline or LEV (i.p.; 50 mg/kg) 1 h before exposure to either normoxia (21% O2; N) or hypoxia (8% O2 of 6 h; H) without reoxygenation. HIF-1α was analyzed by Western blot and immunohistochemistry and mRNA levels were quantified by TaqMan RT-PCR. Hypoxia led to prominent accumulation of cerebral HIF-1α protein in cortical neurons and glial cells and significant up-regulation of VEGF mRNA at P0 (N, 0.018 ± 0.002, vs. H, 0.031 ± 0.003, n = 6; p < 0.05) and P7 (N, 0.096 ± 0.032, vs. H, 0.873 ± 0.069, n = 7; p < 0.001). Interestingly, we detected a significant decrease of iNOS mRNA levels in hypoxic brains. LEV treatment did not alter HIF-1α accumulation either in normoxic or hypoxic brains (P0, P7). Moreover, significant changes of VEGF and NOS mRNA levels did not occur with the exception that hypoxia-induced decreased iNOS levels were not observed in P0 brains. We conclude that acute systemic hypoxia differentially affects expression of HIF-1-regulated vasoactive factors in the newborn mouse brain. Of clinical importance, LEV treatment did not alter crucial HIF-1-regulated neuroprotective mechanisms.

Introduction

Acquired brain lesions as a consequence of perinatal hypoxia are significant causes of neonatal morbidity as well as of severe long-term disability including senso-motor disabilities, postneonatal epilepsy, cognitive and behavioral disturbances (Woodward et al., 2006). Neonatal hypoxic brain injury develops as sequelae of pulmonary immaturity, systemic hypoperfusion, disturbed cerebrovascular autoregulation as well as neonatal complications, e.g. asphyxia, sepsis, apnoic spells and seizures whereas currently no specific neuroprotective treatment is available. The complex hypoxia-induced neurotoxic cascade including excitotoxic, inflammatory and metabolic pathways sustains secondary neurodegeneration during reoxygenation period (Jensen, 2006). Of note, regional and cellular vulnerability to hypoxia is age-specific and determined by distinct developmental factors including paracrine metabolic and vascoactive pathways as well as growth factors (Jensen, 2006). In this context, hypoxia-inducible transcription factor-1 (HIF-1) has been characterized as one of the most important mediators of the molecular response to brain hypoxia as studied in adult (Stroka et al., 2001, Shao et al., 2005) and newborn rodents (Bergeron et al., 2000, Bernaudin et al., 2002). HIF-1 protein is a heterodimer consisting of an α- and β-subunit that belong to the basic-helix–loop–helix (bHLH)/PAS transcription factors. HIF-1 is rapidly degraded by the ubiquitin–proteasome pathway under normoxia. In contrast, hypoxia triggers stabilization and heterodimerization of the HIF-1α:HIF-1β complex that in turn binds to hypoxia response elements of numerous specific HIF-1 target genes modifying oxygen and energy supply, e.g. by activation of vasoproliferative and vasoactive effects (e.g. vascular endothelial growth factor, VEGF; inducible NO synthase, iNOS), glucose utilization and cell survival (Fandrey et al., 2006). The understanding of biological processes influenced by HIF-1 during very early developmental stage due to acute hypoxia is still incomplete. This process is of special interest as efficacy of putative neuroprotective targets is obviously limited to a short immediate time window after hypoxic injury (Bergeron et al., 2000). Whereas mainly protective effects of HIF-1 (Bergeron et al., 2000, Chavez and LaManna, 2002, Grimm et al., 2005, Siddiq et al., 2005) and specific target genes such as VEGF (Jin et al., 2002) or erythropoietin (Grimm et al., 2005) on cell survival have been emphasized, induction of apoptosis due to HIF-1 stabilization under severely hypoxic conditions has been described only recently (Helton et al., 2005). Moreover, neuroprotective as well as toxic effects of the HIF-1-regulated inducible NO synthase isoform (iNOS) have been reported (Golde et al., 2002, Gustavsson et al., 2006, Pannu and Singh, 2006). These observations imply differential regulation of HIF-1-mediated systems on cell survival and cerebral blood flow regulation during impaired oxygenation and reoxygenation altering with developmental age and severity of hypoxic injury (Johnston, 1998, Curristin et al., 2002).

Our study focused on cerebral HIF-1-mediated vasoactive hypoxic stress responses due to acute systemic hypoxia at very early stage of mouse brain development (P0, P7) approximately corresponds to that of the human brain at mid-gestation and near-term, respectively (Dobbing and Sands, 1979).

Apart from hypoxic injury, different antiepileptic drugs are known to enhance vulnerability of the neonatal rodent brain to apoptotic neurodegeneration (Katz et al., 2007). The anticonvulsive drug levetiracetam (LEV; (S)-α-ethyl-2-oxo-pyrrolidine acetamide) that binds to an integral synaptic vesicle membrane protein SV2A (Janz et al., 2003, Lynch et al., 2004) did not exert apoptosis in developing rat brain under normoxic conditions (Manthey et al., 2005). This drug also reduced cellular degeneration in rodent models of focal cerebral hypoxic ischemia (Hanon and Klitgaard, 2001) and epilepsy (Löscher et al., 1998, Marini et al., 2004). These observations suggested neuroprotective properties of LEV. However, data on effects of LEV on hypoxic brain at early developmental stage and the interrelationship between LEV and HIF-1-dependent vasoactive mechanisms are not available.

The purpose of the present study was to characterize HIF-1 protein and specific HIF-1-dependent vasoactive gene responses of the developmental mouse brain at two different developmental stages (P0, P7) to severe short-term systemic hypoxia in the presence or absence of LEV. Additionally, we investigated effects of hypoxia and LEV on hypoxia-inducible NO synthases that are not under the control of HIF-1.

Section snippets

Results

HIF-1α protein, HIF-1-regulated genes as well as hypoxia-inducible but HIF-1-independent vasoactive factors were determined in developing mouse brains at P0 and P7 upon exposure to severe systemic hypoxia with and without LEV treatment.

Discussion

Upon reduced oxygen supply to very young mice (P0 and P7) we observed, firstly, that HIF-1 is rapidly induced in developing cortical neurons. Secondly, we noted that specific vasoactive target genes are differentially affected by this acute hypoxic distress. Thirdly, LEV treatment did not alter accumulation of HIF-1α protein nor the vasoactive HIF-1 target gene VEGF indicating that LEV does not affect crucial endogenous modulators of cerebral adaptation to hypoxia at early stage of brain

Animal experiments

C57BL/6 wild-type mice were exposed to systemic hypoxia (8% O2) without concomitant experimental ischemia at postnatal day 0 (P0, n = 40) and day 7 (P7; n = 38) providing a model for the preterm human neonate at mid-gestation (P0) and the near-term neonate (P7) (Dobbing and Sands, 1979). Duration of hypoxic incubation was 6 h (Hypoxic Workstation INVIVO2 1000, Biotrace International, U.K.). To enable adjustment to the hypoxic environment O2 deprivation was done gradually by decreasing the FiO2 from

Acknowledgments

The authors are grateful to Jessica Braun and Bianca Saam for their skilled technical assistance and to Dr. Urs Ziegler for taking the immunofluorescence pictures. This work was supported by grants from the DFG (Deutsche Forschungsgemeinschaft; R.T.), UCB GmbH (Kerpen, Germany; R.T.) and by the Swiss National Science Foundation (M.G.).

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