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/δVEGFA transgenic mice, glutamate-induced brain lesions are exacerbated suggesting that VEGFA 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 VEGFA had no effect on both apoptotic and necrotic deaths. In contrast, VEGFA abolished the glutamate-induced necrosis observed in layer VI. While MEK and PI3-K inhibitors had no effect on the protective action of VEGFA, L-NAME, a pan inhibitor of NOS, abrogated the effect of VEGFA and exacerbated the excitotoxic action of glutamate. Calcimetry experiments performed on brain slices revealed that VEGFA reduced the massive calcium influx induced by glutamate in layer VI and this effect was blocked by L-NAME. Neuroprotective effect of VEGFA 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 VEGFA.

► In the immature cortex, VEGF prevents necrosis induced by excitotoxic glutamate. ► Protective effect of VEGF is NO-dependent. ► VEGF protection affects GABAergic neurons. ► NO production is initiated by glutamate via regulation of nNOS and NR2B/nNOS coupling. ► In vivo repression of nNOS exacerbates glutamate excitotoxicity.