Neonatal Brain Lesions Research Articles

Perinatal arterial ischemic stroke: how informative is the placenta?

Neuroplacentology is an expanding field of interest that addresses the placental influence on fetal and neonatal brain lesions and on further neurodevelopment. The objective of this study was to clarify the link between placental pathology and perinatal arterial ischemic stroke (PAIS). Prior publications have reported different types of perinatal stroke with diverse methodologies precluding firm conclusions. We report here the histological placental findings in a series of 16 neonates with radiologically confirmed PAIS. Findings were grouped into 3 categories of lesions: (1) inflammation, (2) placental and fetal hypoxic lesions, and (3) placentas with a high birthweight/placenta weight ratio. Matched control placentas were compared to the pathological placentas when feasible. The eight term singleton placentas were compared to a series of 20 placentas from a highly controlled amniotic membrane donation program; in three twin pregnancies, the placental portions from the affected twin and unaffected co-twin were compared. Slightly more than half (9/16, 56%) had histopathological features belonging to more than one category, a feature shared by the singleton control placentas (13/20, 65%). More severe and extensive lesions were however observed in the pathological placentas. One case occurring in the context of SARS-CoV-2 placentitis further expands the spectrum of COVID-related perinatal disease. Our study supports the assumption that PAIS can result from various combinations and interplay of maternal and fetal factors and confirms the value of placenta examination. Yet, placental findings must be interpreted with caution given their prevalence in well-designed controls.

Роль плаценты в развитии поражений головного мозга новорожденного

Роль плаценты в развитии поражений головного мозга новорожденного

Protein S100B and Amplitude-Integrated EEG as Early Predictive Methods for Brain Injury and Seizures in Preterm Neonates

Background: Neonatal brain injury (NBI) is a serious adverse outcome in premature neonates. The most common form of neonatal brain injury (NBI) are intraventricular hemorrhage (IVH) and periventricular leukomalacia (PVL). Improved survival of preterm infants leads to short- and long-term hazards of neurological, cognitive, respiratory, digestive, renal, cardiovascular, metabolic, immune, and psychosocial disturbances. We sought to determine the levels and prognostic value of serum S100B and the role of aEEG during the first three days of life in premature neonates (< 37 weeks) that later developed NBI in the form of intraventricular hemorrhage (IVH) or neonatal seizures to rule out the sensitivity and specificity of them in early detection of brain lesions in preterm neonates.
 Aim of the Study: To evaluate the role of Protein S100B as an early predictor for neonatal brain injury in preterm neonates and the predictive and prognostic value of amplitude-integrated electroencephalography for neonatal brain injury susceptibility and severity.
 Subject and Methods: This study was carried out on 50 preterm neonate (less than 37 weeks GA) who were divided according to presence or absence of brain injury into case and control groups. They were admitted in Neonatal Intensive Care Unit (NICU) of Tanta University Hospital during the period from March 2021 to March 2022.serum S100B at (Day 1 and Day 3), serial trans-cranial sonar and aEEG were done for all patients.
 Results: Neonates with NBI, had significantly higher S100B concentration during the first three days of life, its level was higher in the third day than the first day, the cut-off value >810.4 ng/ml serum S100B performed a sensitivity of 72.7%and a specificity of 71.4% to predict adverse neonatal outcome. 60% neonates had normal aEEG and 40% had abnormal aEEG.72.7% of neonates with NBI had abnormal aEEG interpretation. There was a significant relationship between the electrografic seizures on aEEG and occurrence of clinical seizures as 96.7% of neonates who had abnormal aEEG suffered of clinical convulsion.
 Conclusion: Protein S100B has a good predictive value regarding NBI in premature neonates and aEEG has a great role in monitoring neonatal brain function and early detection of neonatal seizures.

Ophthalmological outcome of 6.5 years children treated for retinopathy of prematurity: a Swedish register study

AimsTo determine the ophthalmological outcome at 6.5 years of age in children treated for retinopathy of prematurity (ROP), and registered in the national Swedish National Register for ROP register.MethodsData on...

The Needed Studies Trying to Untangle the Complex Nature of Neonatal Intracranial Bleeds Occurring around Birth.

The challenge of understanding mechanisms beyond insults and the resulting brain lesions in term or near-term neonates facing delivery time is far from being fully untangled. We believe considering together near-term and term neonates is justified because major achievements in brain maturation have

Fetal and neonatal brain injury in twins complicated by twin anemia polycythemia sequence.

To determine the rate of fetal and neonatal brain lesions and define risk factors for such lesions in pregnancies complicated by Twin Anemia Polycythemia Sequence (TAPS). A retrospective cohort study of monochorionic twin pregnancies which were diagnosed with TAPS in a single tertiary medical center between 2013 and 2021. Pregnancies were followed with fetal brain neurosonogram every 2weeks and fetal brain MRI (magnetic resonance imaging) was performed when indicated at 28-32weeks of gestation; post-natal brain imaging included neonatal brain ultrasound. Pregnancies with pre- and post-natal brain lesions were compared to those without such findings. Overall, 23 monochorionic diamniotic pregnancies were diagnosed with TAPS over the study period resulting in perinatal survival of 91.3% (42/46). In 6/23 (26%) pregnancies and 7/46 (15.2%) fetuses pre- or post-natal brain lesions were detected, of whom five were the polycythemic twins and two were the anemic twins. Brain findings included intra-cerebral hemorrhage and ischemic lesions and were diagnosed prenatally in 6/7 (85.7%) cases. No risk factors for severe brain lesions were identified. TAPS may place the fetuses and neonates at increased risk for cerebral injuries. Incorporation of fetal brain imaging protocols may enhance precise prenatal diagnosis and allow for accurate parental counseling and post-natal care.

Fetal and neonatal brain lesions following laser ablation for twin‐to‐twin‐transfusion‐syndrome as detected by pre‐ and post‐natal brain imaging

To determine the rate of and risk factors for fetal and neonatal brain lesions following laser ablation for twin-to-twin transfusion syndrome (TTTS). A retrospective cohort study of 83 women with monochorionic twin pregnancies who underwent ablation for TTTS at a single tertiary hospital. Post-laser survivors were followed-up with fetal neurosonogram every 2weeks and fetal brain MRI at 28-32weeks of gestation; post-natal brain imaging included neurosonogram. Cases with pre- and post-natal brain lesions were compared to those without. 153 fetuses survived the immediate post-laser period and underwent brain imaging. Of these, 17 (11.11%) exhibited brain lesions on prenatal imaging studies, and 36 (32.4%) on post-natal ultrasound. Later gestational age (GA) at the time of ablation (23.0 vs. 21.4weeks, p=0.0244), post-laser twin-anemia-polycythemia-sequence (TAPS) (29.41% vs. 9.56%, p=0.035) and birthweight discordancy (30% vs. 9%, p=0.0025) were associated with prenatal brain lesions. Earlier GA at delivery (31.0weeks vs. 32.2, p=0.0002) and post-laser TAPS (25% vs. 9.33%, p=0.038) were associated with post-natal brain lesions. Survivors of ablation for TTTS are at risk for brain lesions, which can be detected prenatally. Incorporation of neurosonogram and fetal brain MRI into the routine surveillance of such pregnancies should be considered.

Distinguishing Encephaloclastic Lesions Resulting From Primary or Secondary Pyruvate Dehydrogenase Deficiency From Other Neonatal or Infantile Cavitary Brain Lesions.

The central nervous system (CNS) is a highly complex and energy-dependent organ that is subject to a wide variety of metabolic, hypoxic-ischemic, and infectious insults that result in cystic changes. Diagnosis of metabolic defects causing extensive cystic changes is particularly challenging for the pediatric pathologist, due to the rarity of these conditions. Pyruvate dehydrogenase (PDH) deficiency is one of the most common etiologies of congenital lactic acidosis, caused by mutations in subunits of the large mitochondrial matrix complex, and characterized by periventricular cysts, although few detailed reports focusing on neuropathologic findings exist. In addition, rare defects in other mitochondrial enzymes such as short-chain enoyl-CoA hydratase (SCEH, encoded by ECHS1 gene) can cause secondary PDH deficiency and present with neonatal lactic acidosis, but neuropathological findings have never been reported. Nonmetabolic conditions can also produce CNS cystic lesions, primarily in newborns. The pathologist must therefore distinguish between these etiologically disparate conditions which can produce CNS cavitary lesions. Here, we compare and contrast the gross and microscopic findings of cysts associated with cases of PDH and SCEH deficiencies with other neonatal cystic brain diseases including periventricular leukomalacia, neonatal Alexander disease, Canavan disease, and a case of cysts associated with a vascular abnormality. Our studies show that PDH and SCEH deficiencies are not grossly or histologically distinguishable from each other and both are associated with smooth-walled cysts largely limited to the telencephalic germinal matrix. Both show an absence of prominent hemosiderin deposits, Rosenthal fibers, vacuolization of the white matter, and gliosis or axonal damage in the surrounding parenchyma. These features can help distinguish PDH/SCEH deficiency from other pediatric/neonatal cystic CNS disorders, especially those produced by hypoxic ischemic conditions. Cysts, usually bilateral, confined to the telencephalic germinal matrix should elicit metabolic and genetic testing to appropriately diagnose PDH and SCEH and distinguish them from each other.

Effects of neonatal ethanol on cerebral cortex development through adolescence.

Neonatal brain lesions cause deficits in structure and function of the cerebral cortex that sometimes are not fully expressed until adolescence. To better understand the onset and persistence of changes caused by postnatal day 7 (P7) ethanol treatment, we examined neocortical cell numbers, volume, surface area and thickness from neonatal to post-adolescent ages. In control mice, total neuron number decreased from P8 to reach approximately stable levels at about P30, as expected from normal programmed cell death. Cortical thickness reached adult levels by P14, but cortical volume and surface area continued to increase from juvenile (P20-30) to post-adolescent (P54-93) ages. P7 ethanol caused a reduction of total neurons by P14, but this deficit was transient, with later ages having only small and non-significant reductions. Previous studies also reported transient neuron loss after neonatal lesions that might be partially explained by an acute acceleration of normally occurring programmed cell death. GABAergic neurons expressing parvalbumin, calretinin, or somatostatin were reduced by P14, but unlike total neurons the reductions persisted or increased in later ages. Cortical volume, surface area and thickness were also reduced by P7 ethanol. Cortical volume showed evidence of a transient reduction at P14, and then was reduced again in post-adolescent ages. The results show a developmental sequence of neonatal ethanol effects. By juvenile ages the cortex overcomes the P14 deficit of total neurons, whereas P14 GABA cell deficits persist. Cortical volume reductions were present at P14, and again in post-adolescent ages.

Early Amplitude-Integrated Electroencephalogram as a Predictor of Brain Injury in Newborns With Very Low Birth Weight: A Cohort Study.

To evaluate the relationship between abnormal early amplitude integrated electroencephalography (EEG) and severe lesions in imaging tests performed during the neonatal period in very low birth weight infants. An amplitude-integrated EEG was performed in 70 patients with a mean birth weight of 1226 g during the first 48 hours of life. Severe lesions on magnetic resonance imaging (MRI) or ultrasonography (US) during the neonatal period were considered as adverse conditions. Variables were compared using the χ2 test or analysis of variance. Sensitivity, specificity, and positive likelihood ratio were calculated. Adverse outcomes were observed in 6 patients. There was a significant relationship ( P < .001) between abnormal amplitude-integrated EEG background and severe lesions on MRI and US. Sensitivity and specificity were 100% and 89%, respectively. Early amplitude-integrated EEG with moderate/severe abnormalities in the background is associated with severe structural lesions detected in imaging studies and should be considered as an auxiliary screening tool for the detection of neonatal brain lesions in very low birth weight infants.

Predominant area of brain lesions in neonates with herpes simplex encephalitis.

Nonspecific manifestations and a varied distribution of brain lesions can delay the diagnosis of herpes simplex encephalitis (HSE) in neonates. The aim of this study was to report predominant brain lesions in neonatal HSE, and then to investigate the association between pattern of predominant brain lesions, clinical variables and neurodevelopmental outcome. A multicenter retrospective study was performed in neonates diagnosed with HSE between 2009 and 2014. Magnetic resonance (MR) images, including diffusion-weighted images, were obtained in the acute and chronic phase. Three predominant areas of brain injury could be defined based on characteristic MRI findings in 10 of the 13 infants (77%). The inferior frontal/temporal pole area was involved in five (38%) patients. The watershed distribution was present in six (46%) patients. Four (31%) infants involved the corticospinal tract area. No significant association was found between any predominant distribution of brain lesion pattern and sex, country, viral type or viral load. However, the corticospinal tract involvement was significantly associated with motor impairment (P=0.045). Three predominant areas of brain lesion could be recognized in neonatal HSE. Recognition of those areas can improve prediction of neurodevelopmental outcome.

Cranial ultrasound in detection of neurological lesions in preterm neonates in a tertiary center in North Kerala, India

Background: Incomplete formation and maturation of the central nervous system makes it extremely vulnerable to injury, in the case of premature neonates. This can result in a broad range of neurodevelopmental abnormalities. Cranial ultrasound is a sensitive tool for the early detection of these. Hence the present study was undertaken to assess the prevalence of neurosonological abnormality in preterm infants. The aims of the study were to identify and enumerate the neurosonographic features, to assess the severity of brain injuries by grading the neurosonographic findings and to correlate the clinical presentations with the neurosonographic findings.Methods: The present study was conducted in Department of Radiodiagnosis, Pariyaram Medical College. It consisted of all preterm neonates (less than 37 weeks of gestational age) referred to the Radiology department. The initial scan will be done as soon as possible (within 2 weeks of birth) followed by a repeat scan of the same infants at 36 weeks of corrected age, and at 8weeks post-partum.Results: A total of 100 neonates with gestational age varying from 29 to 37 weeks were studied, with the birth weight varying from 1.5 to 1.9 kg. The most common abnormality found on neurosonogram was germinal-matrix haemorrhage, followed by periventricular leukomalacia.Conclusions: Real time sonography is a sensitive non-invasive initial investigation for the detection of various brain lesions in the preterm neonates.

Brain Injury in Neonates with Complex Congenital Heart Disease: What Is the Predictive Value of MRI in the Fetal Period?

Brain injury in neonates with congenital heart disease is an important predictor of adverse neurodevelopmental outcome. Impaired brain development in congenital heart disease may have a prenatal origin, but the sensitivity and specificity of fetal brain MR imaging for predicting neonatal brain lesions are currently unknown. We sought to determine the value of conventional fetal MR imaging for predicting abnormal findings on neonatal preoperative MR imaging in neonates with complex congenital heart disease. MR imaging studies were performed in 103 fetuses with confirmed congenital heart disease (mean gestational age, 31.57 ± 3.86 weeks) and were repeated postnatally before cardiac surgery (mean age, 6.8 ± 12.2 days). Each MR imaging study was read by a pediatric neuroradiologist. Brain abnormalities were detected in 17/103 (16%) fetuses by fetal MR imaging and in 33/103 (32%) neonates by neonatal MR imaging. Only 9/33 studies with abnormal neonatal findings were preceded by abnormal findings on fetal MR imaging. The sensitivity and specificity of conventional fetal brain MR imaging for predicting neonatal brain abnormalities were 27% and 89%, respectively. Brain abnormalities detected by in utero MR imaging in fetuses with congenital heart disease are associated with higher risk of postnatal preoperative brain injury. However, a substantial proportion of anomalies on postnatal MR imaging were not present on fetal MR imaging; this result is likely due to the limitations of conventional fetal MR imaging and the emergence of new lesions that occurred after the fetal studies. Postnatal brain MR imaging studies are needed to confirm the presence of injury before open heart surgery.

The sigma-1 receptor agonist 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) protects against newborn excitotoxic brain injury by stabilizing the mitochondrial membrane potential in vitro and inhibiting microglial activation in vivo

Premature birth represents a clinical situation of risk for brain injury. The diversity of pathophysiological processes complicates efforts to find effective therapeutic strategies. Excitotoxicity is one important factor in the pathogenesis of preterm brain injury. The observation that sigma-1 receptor agonists possess neuroprotective potential, at least partly mediated by a variety of anti-excitotoxic mechanisms, has generated great interest in targeting those receptors to counteract brain injury. The objective of this study was to evaluate the effect of the highly specific sigma-1 receptor agonist, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) to protect against excitotoxic developmental brain injury in vivo and in vitro. Primary hippocampal neurons were pre-treated with PPBP before glutamate was applied and subsequently analyzed for cell death (PI/calcein AM), mitochondrial activity (TMRM) and morphology of the neuronal network (WGA) using confocal microscopy. Using an established neonatal mouse model we also determined whether systemic injection of PPBP significantly attenuates excitotoxic brain injury. PPBP significantly reduced neuronal cell death in primary hippocampal neurons exposed to glutamate. Neurons treated with PPBP showed a less pronounced loss of mitochondrial membrane potential and fewer morphological changes after glutamate exposure. A single intraperitoneal injection of PPBP given one hour after the excitotoxic insult significantly reduced microglial cell activation and lesion size in cortical gray and white matter. The present study provides strong support for the consideration of sigma-1 receptor agonists as a candidate therapy for the reduction of neonatal excitotoxic brain lesions and might offer a novel target to counteract developmental brain injury.

USE OF DIFFUSION-WEIGHTED MAGNETIC RESONANCE IMAGING FOR REVEALING HYPOXIC-ISCHEMIC BRAIN LESIONS IN NEONATES

The article presents advantages of use of diffusion-weighted magnetic resonance imaging (DW MRI) for revealing hypoxic-ischemic brain lesions in neonates. The trial included 97 neonates with perinatal brain lesion who had been undergoing treatment at a resuscitation department or neonatal pathology department in the first month of life. The article shows high information value of diffusion-weighted images (DWI) for diagnostics of hypoxic-ischemic lesions in comparison with regular standard modes. In the event of no structural brain lesions of neonates, pronounced increase in signal characteristics revealed by DWI indicated considerable pathophysiological alterations. Subsequently, children developed structural alterations in the form of cystic encephalomalacia with expansion of cerebrospinal fluid spaces manifested with pronounced neurological deficit. DW MRI has been offered as a method of prognosticating further neurological development of children on early stages.

Hypoxia-Ischemia or Excitotoxin-Induced Tissue Plasminogen Activator- Dependent Gelatinase Activation in Mice Neonate Brain Microvessels

Hypoxia-ischemia (HI) and excitotoxicity are validated causes of neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor-mediated pathways. Brain microvascular endothelial cells from neonates in culture, contain and release more t-PA and gelatinases upon glutamate challenge than adult cells. We have studied t-PA to gelatinase (MMP-2 and MMP-9) activity links in HI and excitotoxicity lesion models in 5 day–old pups in wild type and in t-PA or its inhibitor (PAI-1) genes inactivated mice. Gelatinolytic activities were detected in SDS-PAGE zymograms and by in situ fluorescent DQ-gelatin microscopic zymographies. HI was achieved by unilateral carotid ligature followed by a 40 min hypoxia (8%O2). Excitotoxic lesions were produced by intra parenchymal cortical (i.c.) injections of 10 µg ibotenate (Ibo). Gel zymograms in WT cortex revealed progressive extinction of MMP-2 and MMP-9 activities near day 15 or day 8 respectively. MMP-2 expression was the same in all strains while MMP-9 activity was barely detectable in t-PA−/− and enhanced in PAI-1−/− mice. HI or Ibo produced activation of MMP-2 activities 6 hours post-insult, in cortices of WT mice but not in t-PA−/− mice. In PAI-1−/− mice, HI or vehicle i.c. injection increased MMP-2 and MMP-9 activities. In situ zymograms using DQ-gelatin revealed vessel associated gelatinolytic activity in lesioned areas in PAI-1−/− and in WT mice. In WT brain slices incubated ex vivo, glutamate (200 µM) induced DQ-gelatin activation in vessels. The effect was not detected in t-PA−/−mice, but was restored by concomitant exposure to recombinant t-PA (20 µg/mL). In summary, neonatal brain lesion paradigms and ex vivo excitotoxic glutamate evoked t-PA-dependent gelatinases activation in vessels. Both MMP-2 and MMP-9 activities appeared t-PA-dependent. The data suggest that vascular directed protease inhibition may have neuroprotection potential against neonatal brain injuries.

Group B Streptococcus Late-Onset Disease: 2003–2010

There is insufficient population-based data on group B streptococcus (GBS) late-onset disease (LOD). Risk factors and routes of GBS transmission are poorly understood. A prospective, cohort study was conducted to collect incidence data on LOD and evaluate GBS infections over an 8-year period (2003-2010). Starting from January 2007, maternal rectovaginal and breast milk cultures were routinely collected on confirmation of the LOD diagnosis to assess maternal GBS culture status. The incidence rate of LOD was 0.32 per 1000 live births (1.4 and 0.24 per 1000 live births for preterm and term newborns, respectively). The registered cases of LOD (n = 100) were classified as sepsis (n = 57), meningitis (n = 36), or focal infection (n = 7). Thirty neonates were preterm (2 had recurrent infection); 68 were term. Four infants died (3 early preterm, 1 term). At the time the LOD diagnosis was confirmed, 3 (6%) of 53 mothers had GBS mastitis, and 30 (64%) of 47 carried GBS at the rectovaginal site. Early (7-30 days) LOD presentation was associated with neonatal brain lesions or death (odds ratio: 0.96 [95% confidence interval: 0.93-0.99]). Intrapartum antibiotic exposure was significantly associated with mild (12 of 22) rather than severe (11 of 45; P = .03) LOD. Preterm neonates had the highest rates of LOD and mortality. Most mothers carried GBS at the time of the LOD diagnosis, whereas 6% had mastitis. Intrapartum antibiotics were associated both with delayed presentation of symptoms and milder LOD.

Use of Human Umbilical Cord Blood Mononuclear Cells to Prevent Perinatal Brain Injury: A Preclinical Study

Cerebral palsy (CP) is the most frequent neurological disorder associated with perinatal injury of the developing brain. Major brain lesions associated with CP are white matter damage (WMD) in preterm infants and cortico-subcortical lesions in term newborns. Cell therapy is considered promising for the repair of brain damage. Human umbilical cord blood mononuclear cells (hUCB-MNCs) are a rich source of various stem cells that could be of interest in repairing perinatal brain damage. Our goal was to investigate the potential of hUCB-MNCs to prevent or repair brain lesions in an animal model of excitotoxic brain injury. We induced neonatal brain lesions using intracranial injections of ibotenate, a glutamate agonist, in 5-day-old rat pups. hUCB-MNCs were injected either intraperitoneally (i.p.) or intravenously (i.v.) soon or 24 h after ibotenate injection, and their neurological effects were assessed using histology and immunohistochemistry. hUCB-MNCs injected i.p. did not reach the systemic circulation but high amounts induced a significant systemic inflammatory response and increased the WMD induced by the excitotoxic insult. This effect was associated with a significant 40% increase in microglial activation around the white matter lesion. hUCB-MNCs injected i.v. soon or 24 h after the excitotoxic insult did not affect lesion size, microglial activation, astroglial cell density, or cell proliferation within the developing white matter or cortical plate at any concentration used. We demonstrated that hUCB-MNCs could not integrate into the developing brain or promote subsequent repair in most conditions tested. We found that the intraperitoneal injection of high amounts of hUCB-MNCs aggravated WMD and was associated with systemic inflammation.

Magnetic resonance imaging versus ultrasound at early post-term age in brain imaging of preterm infants

Objective: To compare the incidence of brain lesions detected on cranial ultrasonography (US) and magnetic resonance imaging (MRI) at early post-term age in a group of preterm infants. Subjects and methods: In this prospective study, entry criteria were very preterm infants with birth weight ≤1250 g or gestational age ≤30 weeks, or brain lesions on early US scan. 73 neonates were included in the study. All infants underwent sequential cranial US scans and an US and MRI scan at early post-term age. Results: Abnormal US and MR findings were detected in 53.4% and 84.9% of neonates respectively. US underestimated the diffuse white matter damage shown in 6.9% and on MRI in 26.4% of neonates, the increase of the subarachnoid space shown on US in 19.2% and on MRI in 43.1% of neonates and thinning of the corpus callosum detected on US in only 5.5% and on MRI in 27.4% of neonates. MRI was the only method to detect abnormalities in the posterior fossa, basal ganglia, cortex and the posterior limb of the internal capsule. US detected with high sensitivity the presence of cystic periventricular leukomalacia, ventricular enlargement, persistent germinal matrix/intraventricular hemorrhage and porencephalic cyst as a result of periventricular hemorrhagic infraction. Conclusion: MRI offers more complete visualization of brain lesions in preterm neonates and is more informative in regard to diagnosis. Follow up assessment of these neonates is needed to show the relationship to neurodevelopment outcome.

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.