Related Experiment Video
Updated: May 26, 2026
The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures
Published on: October 23, 2014
NO-dependent protective effect of VEGF against excitotoxicity on layer VI of the developing cerebral cortex
Faiza El Ghazi1, Arnaud Desfeux, Carole Brasse-Lagnel
1EA 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.
Insights
Vascular Endothelial Growth Factor-A (VEGF-A) protects neonatal mouse brains from glutamate excitotoxicity by activating neuronal nitric oxide synthase (nNOS). This pathway reduces cell death and calcium influx, offering a potential therapeutic target for neonatal brain injury.
Area of Science:
- Neuroscience
- Molecular Biology
- Developmental Biology
Background:
- Cerebral palsy affects infants, with glutamate excitotoxicity causing neonatal brain lesions.
- Previous studies suggest Vascular Endothelial Growth Factor-A (VEGF-A) may protect against excitotoxicity.
Purpose of the Study:
- To investigate the central neuroprotective effects of VEGF-A against glutamate-induced excitotoxicity in neonatal mouse brains.
- To elucidate the underlying molecular mechanisms involving nitric oxide (NO) pathways.
Main Methods:
- Primary cultures of neonatal mouse cortical brain slices exposed to glutamate and VEGF-A.
- Inhibition studies using various enzyme inhibitors (NOS, MEK, PI3-K) and small interfering RNA (siRNA).
- Measurements of cell death (necrosis, apoptosis), calcium influx, NO production, and protein interactions.
Main Results:
- VEGF-A significantly reduced glutamate-induced necrosis in cortical layer VI, without affecting apoptosis.
- The protective effect of VEGF-A was dependent on nitric oxide synthase (NOS) activity, specifically neuronal NOS (nNOS).
- VEGF-A decreased glutamate-induced calcium influx and NO production via nNOS activation, which was neuroprotective in neonates.
Conclusions:
- Glutamate stimulates nNOS activity in neonatal mouse deep cortical layers, leading to neuroprotective NO production.
- VEGF-A exerts its anti-necrotic effect by enhancing this glutamate-induced NO production through nNOS activation.
- This study highlights a novel neuroprotective role of the VEGF-A/nNOS pathway against neonatal excitotoxicity.
Abstract:
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).
