BackgroundHypoxic-ischemic encephalopathy (HIE) is one of the most important causes of neonatal brain injury. Therapeutic hypothermia (TH) is the standard treatment for term newborns after perinatal hypoxic ischemic injury (HI). Despite this, TH does not provide complete neuroprotection. Allopurinol seems to be a good neuroprotector in several animal studies, but it has never been tested in combination with hypothermia.Clinical findings show that male infants with (HI) fare more poorly than matched females in cognitive outcomes. However, there are few studies about neuroprotection taking gender into account in the results.The aim of the present study was to evaluate the potential additive neuroprotective effect of allopurinol when administrated in association with TH in a rodent model of moderate HI. Gender differences in neuroprotection were also evaluated.MethodsP10 male and female rat pups were subjected to HI (Vannucci model) and randomized into five groups: sham intervention (Control), no treatment (HI), hypothermia (HIH), allopurinol (HIA), and dual therapy (hypothermia and allopurinol) (HIHA). To evaluate a treatment’s neuroprotective efficiency, 24 hours after the HI event caspase3 activation was measured. Damaged area and hippocampal volume were also measured 72 hours after the HI event. Negative geotaxis test was performed to evaluate early neurobehavioral reflexes. Learning and spatial memory were assessed via Morris Water Maze (MWM) test at 25 days of life.ResultsDamaged area and hippocampal volume were different among treatment groups (p = 0.001). The largest tissue lesion was observed in the HI group, followed by HIA. There were no differences between control, HIH, and HIHA. When learning process was analyzed, no differences were found. Females from the HIA group had similar results to the HIH and HIHA groups.Cleaved caspase 3 expression was increased in both HI and HIA. Despite this, in females cleaved caspase-3 was only differently increased in the HI group.All treated animals present an improvement in short-term (Negative geotaxis) and long-term (WMT) functional tests. Despite this, treated females present better long-term outcome. In short-term outcome no sex differences were observed.ConclusionsOur results suggest that dual therapy confers great neuroprotection after an HI event. There were functional, histological, and molecular improvements in all treated groups. These differences were more important in females than in males. No statistically significant differences were found between HIHA and HIH; both of them present a great improvement. Our results support the idea of different regulation mechanisms and pathways of cell death, depending on gender.

The cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) gene is expressed from the maternal allele and is located within the centromeric imprinted domain at chromosome 11p15. It is a negative regulator of proliferation, with loss-of-function mutations associated with the overgrowth disorder Beckwith–Wiedemann syndrome. Recently, gain-of-function mutations within the PCNA domain have been described in two disorders characterized by growth failure, namely IMAGe (intra-uterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital abnormalities) syndrome and Silver–Russell syndrome (SRS). Over-expression of CDKN1C by maternally inherited microduplications also results in SRS, suggesting that in addition to activating mutations this gene may regulate growth by changes in dosage. To determine if CDKN1C is involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. We observe higher levels of expression of CDKN1C in IUGR placentas compared to those of controls. All placenta biopsies heterozygous for the PAPA repeat sequence in exon 2 showed appropriate monoallelic expression and no mutations in the PCNA domain were observed. The expression profile was independent of both genetic or methylation variation in the minimal CDKN1C promoter interval and of methylation of the cis-acting maternally methylated region associated with the neighboring KCNQ1OT1 non-coding RNA. Chromatin immunoprecipitation revealed binding sites for CTCF within the unmethylated CDKN1C gene body CpG island and putative enhancer regions, associated with the canonical enhancer histone signature, H3K4me1 and H3K27ac, located ∼58 and 360 kb away. Using 3C-PCR we identify constitutive higher-order chromatin loops that occur between one of these putative enhancer regions and CDKN1C in human placenta tissues, which we propose facilitates expression.

Objective To identify changes in macronutrient content of very preterm human milk associated with perinatal factors. Study design Milk macronutrients were measured on weeks 1, 2, 4 and 8 with mid-infrared transmission spectrometers. Result We assessed 625 samples (from 117 mothers and 130 very preterm infants). Average concentrations were: protein 1.3 ± 0.3 g/dl, carbohydrates 7.3 ± 0.6 g/dl, fat 3.7 ± 1.0 g/dl and energy 296.0 ± 41.0 kJ/dl (70.7 kcal/dl). Gestational age negatively correlated with protein (rho: −0.307, p < 0.001) and energy (r: −0.193, p = 0.003). Advanced maternal age, gestational age and intrauterine growth restriction were independently associated with milk protein content over the first 4 weeks (adjusted R2: 0.113, p = 0.002). Conclusion These findings may help neonatologists identify patients fed Mother´s Own Milk who are at increased risk of poor postnatal growth.