NO-dependent protective effect of VEGF against excitotoxicity on layer VI of the developing cerebral cortex.
In industrialized countries, cerebral palsy affects 2.5‰ of preterm and term infants. At a neurochemical level, the massive release of glutamate constitutes a major process leading to excitotoxicity and neonatal brain lesions. Previous studies, conducted in the laboratory, revealed that, in (δ/δ)VEGF(A) transgenic mice, glutamate-induced brain lesions are exacerbated suggesting that VEGF(A) could play a protective action against excitotoxicity. Using a model of cultured cortical brain slices, the aim of the study was to characterize the central effects of VEGF against glutamate-induced excitotoxicity in neonates. Exposure of brain slices to glutamate induced a strong increase of necrotic cell death in the deep cortical layer VI and a decrease of apoptotic death in superficial layers II-IV. When administered alone, a 6-h treatment with VEGF(A) had no effect on both apoptotic and necrotic deaths. In contrast, VEGF(A) abolished the glutamate-induced necrosis observed in layer VI. While MEK and PI3-K inhibitors had no effect on the protective action of VEGF(A), L-NAME, a pan inhibitor of NOS, abrogated the effect of VEGF(A) and exacerbated the excitotoxic action of glutamate. Calcimetry experiments performed on brain slices revealed that VEGF(A) reduced the massive calcium influx induced by glutamate in layer VI and this effect was blocked by L-NAME. Neuroprotective effect of VEGF(A) was also blocked by LNIO and NPLA, two inhibitors of constitutive NOS, while AGH, an iNOS inhibitor, had no effect. Nitrite measurements, electron paramagnetic resonance spectroscopy and immunohistochemistry indicated that glutamate was a potent inducer of NO production via activation of nNOS in the cortical layer VI. In vivo administration of nNOS siRNA promoted excitotoxicity and mimicked the effects of L-NAME, LNIO and NPLA. A short-term glutamate treatment increased nNOS Ser1412 phosphorylation, while a long-term exposure inhibited nNOS/NR2B protein-protein interactions. Altogether, these findings indicate that, in deep cortical layers of mice neonates, glutamate stimulates nNOS activity. Contrasting with mature brain, NO production induced by high concentrations of glutamate is neuroprotective and is required for the anti-necrotic effect of VEGF(A).
EA NeoVasc 4309, Laboratory of Microvascular Endothelium and Neonate Brain Lesions, Rouen Institute for Biomedical Research, European Institute for Peptide Research (IFR 23), University of Rouen, Rouen, France.
Faiza
F
El Ghazi
Arnaud
A
Desfeux
Carole
C
Brasse-Lagnel
Christian
C
Roux
Celine
C
Lesueur
Danielle
D
Mazur
Isabelle
I
Remy-Jouet
Vincent
V
Richard
Sylvie
S
Jégou
Vincent
V
Laudenbach
Stephane
S
Marret
Soumeya
S
Bekri
Vincent
V
Prevot
Bruno J
BJ
Gonzalez
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D002956:Citrulline / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018683:Excitatory Amino Acid Agents / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D005968:Glutamate Decarboxylase / Q000235:genetics; D018698:Glutamic Acid / Q000633:toxicity; D049452:Green Fluorescent Proteins / Q000235:genetics; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009252:NADPH Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D018683:Excitatory Amino Acid Agents; D018696:Neuroprotective Agents; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002956:Citrulline; D009569:Nitric Oxide; D018698:Glutamic Acid; D026423:S-Nitroso-N-Acetylpenicillamine; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase; D053148:Caspase 3; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; D002118:Calcium