Hypoxic stress responses and levetiracetam: Differential regulation of hypoxia-inducible neuroprotective factors in fetal mouse brain

R. Trollmann; J. Schneider; D. Wenzel; W. Rascher; O. Ogunshola; M. Gassmann

doi:10.1055/s-2008-1079463

Neuropediatrics, Table of Contents

Hypoxic stress responses and levetiracetam: Differential regulation of hypoxia-inducible neuroprotective factors in fetal mouse brain

R Trollmann ¹, J Schneider ¹, D Wenzel ¹, W Rascher ¹, O Ogunshola ², M Gassmann ²

¹Universitätsklinikum, Klinik für Kinder und Jugendliche, Erlangen (D)
²University of Zurich, Center for Integrative Human Physiology, Zürich (CH)

Abstract

Background: Hypoxia-inducible transcription factors (HIFs) have been characterized as the most important mediators of the molecular responses to hypoxia. Experimental stabilization of HIFs and specific target genes, critically involved in vasoactive, metabolic and cytoprotective adaptive mechanisms to hypoxic-ischemic brain lesions, showed neuroprotective effects in adult as well as developing rodent brain. As the antiepileptic drug levetiracetam (LEV) reduces cellular degeneration in focal cerebral ischemia, we aimed to examine its effects on endogenous adaptive factors in developing hypoxic brain. In vivo data on neuroprotective effects of LEV in developing hypoxic CNS are not available in literature.

Objectives: To study 1. effects of LEV on endogenous neurotrophic HIF-α-regulated factors (HIF-1alpha subunit, VEGF, iNOS) in developing mouse brain at different developmental ages (P0, P7), 2. effects of LEV on hypoxia-inducible but HIF-independent NOS isoforms (nNOS, eNOS), and 3. on pro- and anti-apoptotic proteins.

Material and Methods: Fetal C57BL/6 mice (P0, P7) were treated with saline or LEV (i.p.; 50mg/kg) 1h before exposure to normoxia (21% O₂; N) or severe systemic hypoxia (8% O₂, 6h; H). Protein accumulation was investigated by Western blot and immunohistochemistry (cerebral cortex, hippocampus). Gene expression was quantified by TaqMan RT PCR.

Results (mean±SEM): Under normoxia, no significant changes of cerebral HIF-1alpha, VEGF and NOS gene expression were observed in LEV-treated (P0, n=6; P7 n=7) compared to control mice (P0, n=6; P7, n=7). Similarly, HIF-1alpha staining did not show remarkable differences of protein accumulation in hippocampus (HC) or cerebral cortex between saline- and LEV-treated animals. Systemic hypoxia itself led to prominent accumulation of HIF-alpha protein at P0 and P7 as well as significant up-regulation of VEGF at P0 (N, 0.022±0.001, n=6; H, 0.031±0.003, n=6) and at P7 (N, 0.096±0.032, n=7; H, 0.873±0.069, n=7; p<0.001). Interestingly, a significant decrease of iNOS mRNA levels in hypoxic brains at P0 (N, 1.001±0.057; H, 0.733±0.048, p<0.01) and at P7 (N, 1.364±0.083; H, 0.319±0.047, p<0.001) was observed. LEV treatment did not affect HIF-1alpha accumulation in hypoxic cerebral cortex or HC (P0, n=9; P7, n=7) compared to controls (P0, n=6; P7, n=7), or substantially alter gene expression of VEGF, iNOS, eNOS and nNOS, except, a normalization of iNOS decrease in hypoxic P0 brains. We found no influence of LEV treatment on determinants of apoptotic cell death at P0 and P7 assessed by Western blot for anti-apoptotic Bcl-2, Bcl-X_L and pro-apoptotic Bax proteins.

Conclusions: Severe systemic hypoxia differentially affects expression of HIF- regulated vasoactive factors in fetal mouse brain in vivo. Our data indicates that LEV does not induce alterations of crucial endogenous neuroprotective mechanisms of the developing mouse brain. In addition, susceptibility to cerebral apoptosis during acute hypoxia might not be exerted by LEV.