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Neonatal encephalopathy (NE) is a pathological condition affecting long-term neurodevelopmental outcomes. Hypothermia is the only therapeutic option, but does not always improve outcomes; hence, researchers continue to hunt for pharmaceutical compounds. Melatonin treatment has benefitted neonates with hypoxic-ischemic (HI) brain injury. However, unlike animal models that enable the study of the brain and the pathophysiologic cascade, only blood is available from human subjects. Therefore, due to the unavailability of neonatal brain tissue, assumptions about the pathophysiology in pathways and cascades are made in human subjects with NE. We analyzed animal and human specimens to improve our understanding of the pathophysiology in human neonates. A neonate with NE who underwent hypothermia and enrolled in a melatonin pharmacokinetic study was compared to HIrats treated/untreated with melatonin. MicroRNA(miRNA) analyses provided profiles of the neonate's plasma, rat plasma, and rat brain cortexes. We compared these profiles through a bioinformatics tool, identifying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways common to HIbrain injury and melatonin treatment. After evaluating the resulting pathways and the literature, to validate the method, the key proteins expressed in HIbrain injury were investigated using cerebral cortexes. The upregulated miRNAs in human neonate and rat plasma helped identify two KEGG pathways, glioma and long-term potentiation, common to HIinjury and melatonin treatment. A unified neonatal cerebral melatonin-sensitive HI pathwaywas designed and validated by assessing the expression of protein kinase Cα(PKCα), phospho (p)-Akt, and p-ERK proteins in rat brain cortexes. PKCα increased in HI-injured rats and further increased with melatonin. p-Akt and p-ERK returned phosphorylated to their basal level with melatonin treatment after HIinjury. The bioinformatics analyses validated by key protein expression identified pathways common to HIbrain injury and melatonin treatment. This approach helped complete pathways in neonates with NE by integrating information from animal models of HI brain injury.

Purpose Inflammation is a crucial but understudied mechanism of neuronal injury after hypoxia-ischemia. The aim was to identify a panel of cytokines involved in brain injury in neonates with hypoxic ischemic encephalopathy (HIE). Methods Ten newborns with HIE undergoing to therapeutic hypothermia (TH, HIE Group) and 8 healthy newborns (CTRL Group) were enrolled. For the HIE group, 5 samples were collected: between 0 and 6 h of life (time 1), 12 h (time 2), 24 h (time 3), 48 h (time 4) and 96 h of life (time 5). For the CTRL group, one sample was collected. A panel of 48 inflammatory cytokines was determined in all samples. Data were analyzed using multivariate statistical analysis (Principal component analysis, PCA) Results 17 cytokines, among 48 analyzed, were found to be significantly different, initially, between the CTRL and HIE groups: 12 with reported pro-inflammatory effects and 5 with reported anti-inflammatory effects. In the HIE group cytokines showed a decreasing trend during the TH and at the end of treatment comparable to the CTRL group. IL-18 did demonstrate a slight increase at time 3 during HT but decreased steadily at sampling times, 4 and 5. Conclusions Our data demonstrates that many pathways of the inflammatory cascade are activated following hypoxic-ischemic injury. This information will increase our understanding of changes in cytokines over time in neonates with HIE undergoing TH.

The Academy of Breastfeeding Medicine published a clinical protocol for Human Milk storage, recommending refrigeration at a temperature of 4 °C up to 4 d as the optimal conditions for the safety and bactericidal capacity of Human Milk. However, few studies were conducted to evaluate the change in milk composition during this type of refrigeration storage.To elucidate some uncertainties regarding the Human Milk composition and prolonged cold storage, we have investigated the effects of storage at 4 °C up to 96 h on an important category of oxidative stress markers: the Isoprostanes (F2-isoprostanes, F4-neuroprostanes and F3-isoprostanes).The experiment was repeated 3 times to ensure reproducibility of the results. We enrolled 3 donating healthy mothers for each time (total: 9 mothers). Milk was collected with standard extraction methods. Immediately after collection, each Human Milk sample from each mother was pooled and then divided into 5 aliquots. One aliquot (0 h) was immediately frozen at -80 °C until the analysis. The other aliquots (24 h, 48 h, 72 h, 96 h) were stored in a refrigerator at 4 °C respectively for 24, 48, 72 and 96 h, then immediately frozen at -80 °C until the analysis. Milk samples were then used to determine concentration of Isoprostanes in Liquid Chromatography - Mass Spectrometry and Liquid Chromatography - Tandem Mass Spectrometry.Isoprostanes were detectable in all Human Milk samples. There was no significant trend of the concentration of the tested analytes over time.This study provides evidence of the presence in human milk of all the tested isoprostanes: in particular, F2-isoprostanes, F4-neuroprostanes and F3-isoprostanes. Refrigeration and storage of fresh Human Milk in controlled conditions for 96 h did not significantly affect its bioactivity and nutritional quality related with these biomarkers.

Background and Aim: Preterm white matter is vulnerable to lipid peroxidation-mediated injury. F2-isoprostanes (IPs), are a useful biomarker for lipid peroxidation. Aim was to assess the association between early peri-postnatal IPs, white matter injury (WMI) at term equivalent age (TEA), and neurodevelopmental outcome in preterm infants.Methods: Infants with a gestational age (GA) below 28 weeks who had an MRI at TEA were included. IPs were measured in cord blood (cb) at birth and on plasma (pl) between 24 and 48 h after birth. WMI was assessed using Woodward MRI scoring system. Multiple regression analyses were performed to assess the association between IPs with WMI and then with BSITD-III scores at 24 months corrected age (CA). Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive value of pl-IPs for the development of WMI.Results: Forty-four patients were included. cb-IPs were not correlated with WMI score at TEA, whereas higher pl-IPs and lower GA predicted higher WMI score (p = 0.037 and 0.006, respectively) after controlling for GA, FiO2 at sampling and severity of IVH. The area under the curve was 0.72 (CI 95% = 0.51–0.92). The pl-IPs levels plotted curve indicated that 31.8 pg/ml had the best predictive threshold with a sensitivity of 86% and a specificity of 60%, to discriminate newborns with any WMI from newborns without WMI. IPs were not associated with outcome at 24 months.Conclusion: Early measurement of pl-IPs may help discriminate patients showing abnormal WMI score at TEA, thus representing an early biomarker to identify newborns at risk for brain injury.

Chemotherapy for castration-resistant prostate cancer (CRPC) is only temporarily effective due to the onset of chemoresistance. We investigated the efficacy of NO- and H2S-releasing doxorubicins (NitDox and H2SDox) in overcoming drug resistance and evaluated their safety. New and innovative NO- and H2S-releasing doxorubicins (NitDox and H2SDox) showed a good intracellular accumulation and high cytotoxic activity in vitro in an androgen-independent and doxorubicin-resistant DU-145 prostate cancer cell line. Nude mice were subcutaneously injected with 4*106 DU-145 cells and treated once a week for 3 weeks with 5 mg/kg doxorubicin, NitDox, H2SDox or vehicle, i.p. Animal weight, tumor volume, intra-tumoral drug accumulation, apoptosis and the presence of nitrotyrosine and sulfhydryl (SH) groups within the tumor, were evaluated. Cardiotoxicity was assessed by measuring troponin plasma levels and the left ventricular wall thickness. In vivo, NitDox and H2SDox accumulated inside the tumors, significantly reduced tumor volumes by 60%, increased the percentage of apoptotic cells in both the inner and the outer parts of the tumors and the presence of nitrotyrosine and SH groups. Doxorubicin treatment was associated with reduced body weight and cardiotoxicity. On the contrary, NitDox and H2SDox were well tolerated and had a better safety profile. Combining efficacy with reduced cardiovascular side effects, NitDox and H2SDox are promising novel therapeutic agents for reversing chemoresistance in CRCP.

Polyunsaturated fatty acid damages lead to alterations in sperm function. This study aimed to investigate the involvement of F2-isoprostanes (F2-IsoPs), oxidized lipid products from arachidonic acid, in sperm quality impairment. For this purpose, F2-IsoP levels in semen and F2-IsoP localization in spermatozoa were explored in infertile subjects affected by idiopathic infertility or varicocele, as well as in fertile men. As compared to fertile men, in the idiopathic infertility and varicocele groups, sperm concentration, motility, morphology, viability, and fertility index were significantly lower and the mean scores concerning sperm apoptosis, necrosis, and immaturity were significantly higher. The idiopathic infertile group showed a reduction in sperm motility and fertility index, as well as an increase of apoptosis and necrosis percentages, in comparison to the varicocele group. The varicocele group showed the highest levels of F2-IsoPs, a significant increase of sperm immaturity, and a significant correlation between F2-IsoP levels and sperm immaturity. 8-Iso Prostaglandin F2α, biomarker of in vivo F2-IsoP, was clearly localized in sperm midpiece and cytoplasmic residues. Data show that F2-IsoP formation is relevant in semen and sperm from infertile patients with varicocele and high percentage of immaturity, suggesting that a correct fatty acid integrity is needed for sperm maturation.

BackgroundEarly-onset neonatal sepsis (EOS) is a serious and potentially life-threatening disease in newborns. C reactive protein (CRP) is the most used laboratory biomarker for the detection of EOS. Little is known about normal reference values of CRP during the perinatal period as several factors are able to influence it.ObjectivesTo identify an appropriate range of CRP values in healthy term newborns during the first 48 hours of life.DesignCRP determination was performed in 859 term newborns at 12, 24 and 48 hours of life. Mode of delivery, maternal vaginal culture results, intrapartum antimicrobial prophylaxis (IAP) and other perinatal variables were recorded.ResultsCRP mean values were significantly higher at 48 hours (4.10 mg/L) than at both 24 (2.30 mg/L) and 12 hours of life (0.80 mg/L). CRP levels were affected by a number of perinatal proinflammatory variables. In particular, CRP mean values were significantly higher in babies born by vaginal delivery (3.80 mg/L) and emergency caesarean section (3.60 mg/L) than in babies born by elective caesarean section (2.10 mg/L). Completed course of IAP led to lower CRP mean values (2.90 mg/L) than IAP not completed (3.80 mg/L) or not performed (4.70 mg/L).ConclusionsPostnatal age and mode of delivery significantly influence CRP values. Reliable reference values are crucial in order to obtain an adequate diagnostic accuracy.

This research paper aims to investigate if oxidative stress biomarkers increase after a painful procedure in term newborns and if nonpharmacological approaches, or sex, influence pain degree, and the subsequent OS. 83 healthy term newborns were enrolled to receive 10% oral glucose or sensorial saturation (SS) for analgesia during heel prick (HP). The ABC scale was used to score the pain. Advanced oxidation protein products (AOPP) and total hydroperoxides (TH) as biomarkers of OS were measured at the beginning (early-sample) and at the end (late-sample) of HP. The early-sample/late-sample ratio for AOPP and TH was used to evaluate the increase in OS biomarkers after HP. Higher levels of both AOPP and TH ratio were observed in high degree pain (4–6) compared with low degree pain score (0–3) (AOPP:p=0.049; TH:p=0.001). Newborns receiving SS showed a significantly lower pain score (p=0.000) and AOPP ratio levels (p=0.021) than those without. Males showed higher TH levels at the end of HP (p=0.005) compared to females. The current study demonstrates that a relationship between pain degree and OS exists in healthy full-term newborns. The amount of OS is gender related, being higher in males. SS reduces pain score together with pain-related OS in the newborns.

Oxidative stress (OS) is a common pathogenic factor involved in the onset of several diseases in humans, from immunologic disorders to malignancy, being a serious public health problem. In perinatal period, OS has been associated with adverse outcome of pregnancy and neonatal diseases. Dangerous effects of OS are mediated by increased production of free radicals (FRs) following various mechanisms, such as hypoxia, ischemia reperfusion, hyperoxia, inflammation, mitochondrial dysfunction, Fenton chemistry, and prostaglandin metabolism. FRs have short half-life, and their measurement in vivo is faced with many challenges. However, oxyradical derivatives are stable and thus may be measured and monitored repeatedly. The quantification of OS is based on the measurement of specific biomarkers in biologic fluids and tissues, which reflect induced oxidative damage to lipids, proteins, and DNA. Prostanoids, non–protein-bound iron (NPBI), and advanced oxidation protein products (AOPP) are actually considered truly specific and reliable for neonatal injury. Defining reference values for these biomarkers is necessary to investigate their role in neonatal diseases or also to evaluate the success of treatments. In this work, we wanted to define laboratory reference values for biomarkers of OS in a healthy population of term newborns.

The aim of this paper was to investigate the relevance of isoprostanoids i.e., F

Background From fetal life until cardiac surgery, complex congenital heart diseases (CHD) exhibit different hemodynamic and oxygenation patterns that can lead to alteration of the metabolic profile. We used a metabolomic approach to identify urine metabolic markers before cardiac surgery, aiming to define the physiology of patients with complex CHD and to contribute to predict their neurodevelopmental outcome. Methods In a prospective, observational, single-center study we enrolled 28 patients with complex biventricular and univentricular CHD aged less than 5 years, on stable hemodynamic conditions, and with no genetic anomalies. We analyzed urine samples, collected at the induction of anesthesia, by 1H NMR spectroscopy. Profiles of 1H NMR spectra were submitted to unsupervised (principal component) and supervised (partial least squares-discriminant) multivariate analysis. Neurodevelopment was assessed by neuropsychological and adaptive functioning testing. Results Principal components analysis divided CHD patients metabolic profiles in two distinct clusters (RED and BLACK). Metabolic profiles belonging to the RED cluster showed higher levels of accumulation of citric acid cycle intermediates and glucose compared to the profiles in the BLACK cluster, indicating a possible switching to anaerobic metabolism. Patients belonging to the RED cluster were significantly more prone to show an adverse neurodevelopment pattern (p = 0.01). Conclusions The application of metabolomic analysis to CHD children permitted a deeper insight on their metabolic status that could help to obtain a better understanding of the physiological implications and to predict long-term neurodevelopmental outcome.

Melatonin is both a potent free radical (FR) scavenger and a broad-spectrum antioxidant. It seems to give useful effects in newborn disorders as has been shown for adults. The unbalance between prooxidant and antioxidant factors leads to oxidative stress (OS) and damage to biomolecules. OS is involved in the pathogenesis of many chronic diseases in adulthood, such as atherosclerosis, cancer, diabetes, rheumatoid arthritis, stroke and postischemic perfusion injury, myocardial infarction and cardiovascular diseases, chronic inflammation, septic shock, aging, and other degenerative diseases Nevertheless, there is growing evidence that OS is involved in the pathogenesis of many fetal and newborn diseases. The unbalance between a low-efficient antioxidant system and an overproduction of FR, especially in preterm babies, leads to the so-called FR-related disease of newborns, characterized by several cellular, tissue, and organ damage (kidney, retina, lung, bowel, and brain injury). Among antioxidants, melatonin (MLT) shows high antioxidant and anti-inflammatory properties: it is able to scavenge dangerous FR; it induces the production of antioxidant enzymes; it has no prooxidant effects; and it is safe. During the last decade, MLT has started to be considered as an attractive option to minimize as much as possible the sequelae from OS damage: in damaged lung tissue, MLT attenuates the hyperoxia-induced depletion of antioxidant enzyme activities and reduces proinflammatory cytokines; in animal model affected with necrotizing enterocolitis (NEC), MLT reduces tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β), lipid peroxidation products, reactive oxygen species/reactive nitrogen species (ROS/RNS), and it reverses lipopolysaccharide-induced motility disturbances; in developing retina, MLT prevents retinal ganglion cell death through its antioxidant and anti-inflammatory properties. In particular, MLT appears as a very interesting drug to reduce the neurological sequelae from hypoxic-ischemic brain injury. Because of its lipophilic properties, MLT easily crosses most biological cell membranes, including the placenta and the blood–brain barrier and may prevent neonatal brain injury in early stage of life. The neuroprotective role of MLT calls for further investigation in the newborn infants.

Fetal programming occurs when the normal pattern of fetal development is disrupted by an abnormal stimulus or an “insult” during intrauterine life, which leads to adaptations by the fetus to allow its survival but could finally result in permanent structural and physiological changes with long-term consequences in adulthood. The availability of nutrients, hormones, and respiratory gases is the principal determinant of fetal growth and offspring's subsequent health. Fetal nutrient and oxygen availability depend on the rate of transfer across the “placental barrier.” Nutritional status of the mother is also important: both maternal undernutrition and/or overnutrition during early gestation may increase the incidence of cardiovascular and metabolic disorders in the offspring in later life. Oxidative stress has been supposed to be the link between adverse intrauterine environment and later elevated risks of chronic diseases. It is an important initiating mechanism underlying the programming process due to suboptimal nutrition. Antioxidant vitamins, proteins, and trace elements can be compromised under condition of poor maternal nutrition leading to oxidant/antioxidant imbalance during pregnancy. On the other hand, maternal overnutrition is associated to chronic inflammatory states that increase free radicals' production. Developing dietary strategies to optimize maternal nutrition is necessary to supply the fetus with appropriate substrates and to avoid fetal redox status disruption.

Intraventricular hemorrhage (IVH) in very preterm infants is a common disease, which can induce long-term consequences. Oxidative stress (OS) occurs easily in preterm newborns due to an imbalance between high free radical (FR) production and the low antioxidant shield, not completely developed at birth. Nonprotein-bound iron (NPBI) concentration in cord blood has been found to be highly predictive for the risk of poor neurodevelopmental outcome. However, at present, no data exist about the exact mechanisms associated with IVH induced by iron-mediated FRs. We propose the hypothesis that hypoxia or ischemia-induced releasing of NPBI is a key regulating event that initiates a vicious circle of excessive FR generation, which in turn participates in edema development, inflammatory reaction, and endothelial injury. This suggests that developing effective neuroprotective strategies for preterm infants requires a detailed understanding of OS reactions and glial responses.

La Sindrome delle Apnee Ostruttive Notturne (OSAS) è una patologia caratterizzata da alterazioni metaboliche e da un elevato rischio di sviluppo di patologie cardiovascolari. Lo scopo dello studio è stato quello di identificare dei markers precoci predittivi di rischio cardiovascolare con la valutazione dello stress ossidativo misurato attraverso esami di laboratorio in soggetti normali e pazienti con diagnosi di sindrome delle apnee ostruttive notturne. È stato effettuato uno studio prospettico per confrontare i risultati di laboratorio ottenuti dalla valutazione dei biomarkers dello stress ossidativo in 20 pazienti adulti con OSAS e 20 soggetti sani. Le tecniche di analisi utilizzate avevano l’obiettivo di identificare e quantificare i danni dei radicali liberi attraverso la misurazione di anti-ossidanti e pro-ossidanti in modo da valutare l’equilibrio ossidativo presente nei due gruppi di studio. I due gruppi di pazienti sono risultati omogeni per sesso, età ed indice di massa corporea (p < 0,05). Una differenza statisticamente significativa è stata individuata tra i livelli di indice di apnea-ipopnea valutata alla polisonnografia e di isoprostani, produzione di proteine di ossidazione e proteine non legate al ferro nei due gruppi in esame. Nessuna differenza significativa è stata trovata nel livello dei tioli tra i soggetti sani e i pazienti con sindrome delle apnee ostruttive. I tioli, a differenza degli altri markers, sono molecole anti-ossidanti, i restanti sono invece espressione di danno ossidativo. I risultati dello studio indicano che i biomarkers potrebbero essere utilizzati come indici di ostruzione delle vie aeree superiori (VAS) e come marcatori precoci di ipossiemia causando processi flogistici ricorrenti e danno locale da radicali liberi a carico delle VAS.

The aim of this paper was to investigate the relevance of isoprostanoids i.e., F2-isoprostanes (F2-IsoPs), F4-neuroprostanes (F4-NeuroPs) and F2-dihomo-isoprostanes (F2-dihomo-IsoPs) in semen quality. Isoprostanoid levels were detected in semen of fertile and infertile men with varicocele or idiopathic infertility. Semen quality was assessed by light microscopy and transmission electron microscopy; the relationships between isoprostanes and semen parameters were also explored. F2-IsoPs levels were significantly different in the varicocele group compared to idiopathic infertile group and fertile men (P < 0.01 and P < 0.001 respectively). Moreover, F2-dihomo-IsoP values were significantly higher in varicocele group respect to fertile men (P < 0.05). No significant statistical differences were found regarding F4-NeuroP concentrations. In the whole population, F2-IsoPs positively correlated with F2-dihomo-IsoPs and both isoprostanoids showed a positive correlation with immaturity and a negative correlation with sperm motility. F2-IsoP levels were positively correlated with the percentage of immaturity in infertile varicocele groups (P < 0.01) whereas a significant relationship between F4-NeuroP values and the percentage of sperm necrosis was shown in idiopathic infertility group (P < 0.01). A significant negative correlation of F4-NeuroPs with sperm morphology was detected in infertile varicocele subjects (P < 0.05). This study suggests that isoprostanoid semen levels appear to be associated with male infertility being related to the sperm quality and confirming the important role of fatty acids profiling in human sperm maturation.

Background Pregnancy is characterized by multiple metabolic processes to allow proper foetal development and ensure adequate stores. Little is known about the interactions between maternal and foetal metabolism during the last phase of pregnancy. Metabolomic offers potential to discover changes in maternal metabolism in pregnancy and their relation to the newborn metabolic status. Objective In this study we tested the hypothesis that metabolomic status in newborns at birth depends upon the metabolomic profile of their mothers in the last phase of pregnancy. Study design Urine samples were collected from 36 pregnant women three weeks before delivery and from 21 healthy term newborns within 48 h after birth. Urines were analysed using proton nuclear magnetic resonance (1H NMR) spectroscopy and NMR urine spectra were evaluated through Principal Components Analysis. Results The first component of the PCA analysis showed two distinct metabolic groups: pregnant women and newborns. A significant correlation was found between urine metabolic profiles of newborns and those of their mothers. Conclusion Urine metabolomic profiles of newborns at birth mirrors that of their mothers in the last phase of pregnancy. The metabolomic approach appears to be crucial to understand the maternal effects on foetal programming and infant outcomes.

Objectives The composition of milk from mothers delivering prematurely differs from that of full-term mature milk and changes over time. The aim of this study is to test the hypothesis that changes in milk metabolomic profile from mothers delivering prematurely persist over time when compared with mothers delivering at term. Methods Nuclear magnetic resonance spectroscopy was used to analyze the metabolome pattern of human milk samples collected from 18 mothers. Twelve mothers collected 12 term milk samples (one for each mother) once between 4 and 7 d after delivery. Six mothers delivering prematurely (29–31 wk of gestational age) and collected three samples each, once a week after delivery until the third week after birth. Results Principal component analysis identified two distinct metabolite groups, one represented by the 18 preterm milk samples and the other by term milk samples. Metabolite profiling identified that lactose and oligosaccharide levels were significantly more represented in preterm than in milk term samples. Conclusions The preterm milk metabolome pattern undergoes maturation during the first 3 wk after birth, but at the end of the third week it still does not resemble the term milk pattern. The specific changes in mothers’ milk metabolomic profiles according to their offspring might reflect the different nutritional requirement of each preterm infant. This knowledge is crucial to move from standardized nutritional protocols to tailored, individualized nutrition in preterm infants.

This research paper is aimed at evaluating the predictive role of a default panel of oxidative stress (OS) biomarkers for the early identification of infants at high risk of HIE and their validation through the correlation with MRI findings. A multicenter prospective observational study was performed between March 2012 and April 2015 in two European tertiary NICUs. Eighty-four term infants at risk for HIE (pH ′ Apgar ′ Apgar score, pH, and BE and higher Thompson score were found in group II compared to group I at birth. Group II showed significantly higher AOPP and NPBI levels than group I (mean (SD) AOPP: 15.7 (15.5) versus 34.1 (39.2), p=0.033; NPBI 1.1 (2.5) versus 3.9 (4.4), p=0.013) soon after birth (P1). No differences were observed in OS biomarker levels between the two groups at P2 and P3. A regression model, including adjustment for hypothermia treatment, gender, and time after birth, showed that AOPP levels and male gender were both risk factors for higher brain damage scores (AOPP: OR 3.6, 95% CI (1.1–12.2) and gender: OR 5.6, 95% CI (1.2–25.7), resp.). Newborns with severe asphyxia showed higher OS than those with mild asphyxia at birth. AOPP are significantly associated with the severity of brain injury assessed by MRI, especially in males.

Background and Objective. Early identification of neonates at risk for brain injury is important to start appropriate intervention. Urinary metabolomics is a source of potential, noninvasive biomarkers of brain disease. We studied the urinary metabolic profile at 2 and 10 days in preterm neonates with normal/mild and moderate/severe MRI abnormalities at term equivalent age.Methods. Urine samples were collected at two and 10 days after birth in 30 extremely preterm infants and analyzed using proton magnetic resonance spectroscopy. A 3 T MRI was performed at term equivalent age, and images were scored for white matter (WM), cortical grey matter (cGM), deep GM, and cerebellar abnormalities. Infants were divided in two groups: normal/mild and moderately/severely abnormal MRI scores.Results. No significant clustering was seen between normal/mild and moderate/severe MRI scores for all regions at both time points. The ROC curves distinguished neonates at 2 and 10 days who later developed a markedly less mature cGM score from the others (2 d: area under the curve (AUC) = 0.72, specificity (SP) = 65%, sensitivity (SE) = 75% and 10 d: AUC = 0.80, SP = 78%, SE = 80%) and a moderately to severely abnormal WM score (2 d: AUC = 0.71, specificity (SP) = 80%, sensitivity (SE) = 72% and 10 d: AUC = 0.69, SP = 64%, SE = 89%).Conclusions. Early urinary spectra of preterm infants were able to discriminate metabolic profiles in patients with moderately/severely abnormal cGM and WM scores at term equivalent age. Urine spectra are promising for early identification of neonates at risk of brain damage and allow understanding of the pathogenesis of altered brain development.

During the perinatal period, free radicals (FRs) are involved in several physiological roles such as the cellular responses to noxia, the defense against infectious agents, the regulation of cellular signaling function, and the induction of a mitogenic response. However, the overproduction of FRs and the insufficiency of an antioxidant mechanism result in oxidative stress (OS) which represents a deleterious process and an important mediator of damage to the placenta and the developing fetus. After birth, OS can be magnified by other predisposing conditions such as hypoxia, hyperoxia, ischemia, hypoxia ischemia-reperfusion, inflammation, and high levels of nonprotein-bound iron. Newborns are particularly susceptible to OS and oxidative damage due to the increased generation of FRs and the lack of adequate antioxidant protection. This impairment of the oxidative balance has been thought to be the common factor of the so-called “free radical related diseases of prematurity,” including retinopathy of prematurity, bronchopulmonary dysplasia, intraventricular hemorrhage, periventricular leukomalacia, necrotizing enterocolitis, kidney damage, and oxidative hemolysis. In this review, we provide an update focused on the factors influencing these diseases refining the knowledge about the role of OS in their pathogenesis and the current evidences of such relationship. Mechanisms governing FR formation and subsequent OS may represent targets for counteracting tissue damage.

Background. Rooming-in practice improves breastfeeding and reduces newborn stress reactivity. When this modality is not available, partial rooming-in after birth can be considered. Salivary cortisol levels (SCLs) are considered reliable biomarkers to indicate stress. Objective. To test the hypothesis that rooming-in duration impacts neonatal stress response in hospitalized newborns. Design/methods. Forty term newborns, enrolled in the Neonatology and Obstetrics Nursing, C.G. Ruesch, Naples, Italy, were divided, according to the mother’s choice, into the study (SG; n=20) and control (CG; n=20) groups if they received full (24 hs) or partial (14 hs) rooming-in care, respectively. Saliva samples were collected from all babies between 7:00 a.m. and 8:00 a.m. of the 3rd day of life by using oral swab. Salivary cortisol levels were measured using an enzyme immunoassay kit (Salimetrics LLC, PA, USA). Results. A statistically significant difference in the SCLs between SG and CG was found (median: 258 ng/dl versus 488.5 ng/dl; p=0.048). Conclusions. Data support the practice of full rooming-in care compared with partial rooming-in. The rooming-in duration clearly reduces SCLs and likely neonatal stress. These lower SCLs may have long-term positive effects reducing the risk of metabolic syndrome, high blood pressure, and cognitive and behavioural changes.

Melatonin possesses potential efficacy in perinatal brain injuries, and has been proposed as adjunctive pharmacological therapy in combination with hypothermia in the clinical setting. However, the pharmacokinetics of melatonin in preterm and term newborns is still unknown. The aim of this study was to analyze the pharmacokinetics of melatonin after intragastric administration in preterm infants. Preterm newborns were enrolled 24–72 h after birth, and randomly assigned to three groups receiving a single bolus of 0.5 mg·kg−1 melatonin, or 3 boluses of 1 or 5 mg·kg−1 of melatonin at 24-h intervals. Blood samples were collected before and at selective times after melatonin administration. The half-life of melatonin in plasma ranged from 7.98 to 10.94 h, and the area under the curve (AUC) from 10.48 to 118.17 µg·mL−1·h−1. Our results indicate a different pharmacokinetic profile in premature newborns, compared to adults and experimental animals. The high peak plasma concentrations and the long half-life indicate that in the neonatal clinical setting, it is possible to obtain and maintain high serum concentrations using a single administration of melatonin repeated every 12/24 h.

Background: Perinatal hypoxic-ischemic brain damage is a major cause of acute mortality and chronic neurological morbidity in infants and children. Oxidative stress due to free radical formation and the initiation of abnormal oxidative reactions appears to play a key role. Docosahexanoic acid (DHA), a main component of brain membrane phospholipids, may act as a neuroprotectant after hypoxia-ischemia by regulating multiple molecular pathways and gene expression. Objectives: The aims of this study were to test the hypothesis that DHA provides significant protection against lipoperoxidation damage in the cerebral cortex and hippocampus in a neonatal piglet model of severe hypoxia-reoxygenation. Methods: Newborn piglets, Noroc (LYLD), were subjected to severe global hypoxia. One group was resuscitated with ambient air (21% group, n = 11) and another also received 5 mg/kg of DHA 4 h after the end of hypoxia (21% DHA group, n = 10). After 9.5 h, tissues from the prefrontal cortex and hippocampus were sampled and the levels of isoprostanes, neuroprostanes, neurofurans, and F2-dihomo-isoprostanes were determined by the liquid chromatography triple quadrupole mass spectrometry technique. Results: Lipid peroxidation biomarkers were significantly lower in both the cortex and hippocampus in the DHA-treated group compared with the untreated group. Conclusions: The present study demonstrates that DHA administration after severe hypoxia in newborn piglets has an antioxidative effect in the brain, suggesting a protective potential of DHA if given after injuries to the brain.

Premesse . Il dosaggio della glicemia, effettuato nei Laboratori clinici, risulta associato a diverse problematiche. Per esempio la permanenza del campione in vitro, oltre alla leucocitosi e alla contaminazione batterica, favorisce la glicolisi con conseguente diminuzione della concentrazione di glucosio, stimata in circa il 7% per ogni ora che intercorre dal momento in cui e stato effettuato il prelievo ematico al dosaggio. A tale proposito risulta necessario migliorare la fase preanalitica in modo da uniformare e ottimizzare i dosaggi della glicemia. Degli accorgimenti risolutivi potrebbero essere la separazione precoce delle cellule o un raffreddamento veloce del campione, anche se risultano difficili da mettere in pratica. Il fluoruro noto come anticoagulante e anche un inibitore glicolitico, per cui il suo utilizzo, nel dosaggio glicemico, ha permesso una riduzione del problema, ma non la risoluzione, in quanto il fluoruro agisce solo a valle della glicolisi, precisamente sull’enzima enolasi. L’obiettivo e trovare sostanze inibenti la glicolisi che agiscano a monte di tale processo, annullando o riducendo il consumo di glucosio al minimo nella fase pre-analitica. Il citrato, comunemente utilizzato in Laboratorio come anticoagulante, ha la proprieta di agire nelle prime tappe della glicolisi inibendo l’esochinasi e la fosfofruttochinasi.

Oxidative stress plays a key role in carcinogenesis. Oxidative damage to cell components can lead to the initiation, promotion and progression of cancer. Oxidative stress is also a distinctive sign in several genetic disorders characterized by a cancer predisposition such as ataxia-telangiectasia, Fanconi anemia, Down syndrome, Beckwith-Wiedemann syndrome and Costello syndrome. Taking into account the link between oxidative stress and cancer, the capacity of antioxidant agents to prevent or delay neoplastic development has been tested in various studies, both in vitro and in vivo, with interesting and promising results. In recent years, research has been conducted into the molecular mechanisms linking oxidative stress to the pathogenesis of the genetic syndromes we consider in this review, with the resulting identification of possible new therapeutic targets. The aim of this review is to focus on the oxidative mechanisms intervening in carcinogenesis in cancer-prone genetic disorders and to analyze the current status and future prospects of antioxidants.

Objectives. To test the hypothesis that neonatal supplementation with lutein in the first hours of life reduces neonatal oxidative stress (OS) in the immediate postpartum period.Methods. A randomized controlled, double-blinded clinical trial was conducted among 150 newborns divided into control group, not supplemented (n=47), and test group, supplemented with lutein on the first day postpartum (n=103). Blood Samples were collected at birth from cord and at 48 hrs postpartum while routine neonatal metabolic screenings were taking place. Total hydroperoxide (TH), advanced oxidation protein products (AOPP), and biological antioxidant potential (BAP) were measured by spectrophotometry and data were analyzed by Wilcoxon rank sum test and by multivariate logistic regression analysis.Results. Before lutein supplementation, the mean blood concentrations of AOPP, TH, and BAP were 36.10 umol/L, 156.75 mmol/H2O2, and 2361.04 umol/L in the test group. After lutein supplementation, significantly higher BAP increment (0.17 ± 0.22 versus 0.06 versus ± 0.46) and lower TH increment (0.46 ± 0.54 versus 0.34 ± 0.52) were observed in the test group compared to controls.Conclusion. Neonatal supplementation with lutein in the first hours of life increases BAP and reduces TH in supplemented babies compared to those untreated. The generation of free radical-induced damage at birth is reduced by lutein. This trial is registered with ClinicalTrials.govNCT02068807.

Summary We performed a randomized study in preterm lambs to assess the hypothesis that treatment with a natural surfactant combined with superoxide dismutase (SOD) and catalase (CAT) might decrease pulmonary oxidative stress in an animal model of respiratory distress syndrome (RDS). Animals received 200 mg/kg of porcine natural surfactant or 200 mg/kg of natural surfactant combined with 2 mg/ml of SOD and 3,000 U/ml of CAT. Lung tissue oxidation was studied by measuring total hydroperoxide (TH), advanced oxidation protein products (AOPP), and non-protein bound iron (NPBI) in bronchial aspirate samples. In addition, the animal's lung mechanics were evaluated. TH, AOPP, and NPBI were lower in the groups treated with surfactant plus SOD and CAT than in the surfactant group, while lung mechanics did not vary. We concluded that natural surfactant combined with SOD and CAT is effective in reducing the oxidative lung stress in an animal model of RDS. Pediatr Pulmonol. 2014; 49:898–904. © 2013 Wiley Periodicals, Inc.

Obstructive sleep apnoea syndrome (OSAS) is a sleep disorder that leads to metabolic abnormalities and increased cardiovascular risk. This study aimed to define the expression and clinical significance of biomarkers involved in oxidative stress in patients with OSAS. A prospective study was designed to compare outcomes of oxidative stress laboratory tests in three groups of subjects. The study involved the recruitment of three groups of subjects, 10 patients with obstructive sleep apnoea syndrome with AHI30; 10 patients suffering from snoring at night with AHI15; 10 patients with nasal respiratory impairment with AHI5. Patients were subjected to skin prick tests for common aero-allergens, nasal endoscopy, active anterior rhinomanometry, fibrolaryngoscopy and polysomnography; and extra-routine diagnostic tests and procedures; analysis of oxidative and antioxidant (plasma thiol groups) biomarkers in blood and urine samples. No statistical differences in age, sex distribution or body mass index were present between the three groups (p0.05). There were significant differences in AHI among the three groups of patients (p0.05). No statistical significance was found in the Analysis of Variance (ANOVA) test (p0.05) between the levels of biomarkers of oxidative stress in the three populations studied. The results of our study show that the nose can play a role in the pathogenesis of OSAS through the production of biomarkers of oxidative stress.La sindrome delle apnee ostruttive del sonno (OSAS) è una malattia che può portare ad alterazioni metaboliche e a un’aumentata incidenza di patologie cardiovascolari. Questo studio ha lo scopo di definire l’espressione e il significato clinico di biomarkers coinvolti nello stress ossidativo nei pazienti con diagnosi di OSAS. I risultati degli esami di laboratorio dello stress ossidativo sono stati confrontai prospetticamente in tre gruppi di soggetti: 10 con sindrome delle apnee ostruttiva del sonno con Apnea Hypopnea Index (AHI)30; 10 con roncopatia notturna e AHI15 e 10 con insufficienza respiratoria nasale e AHI5. I pazienti sono stati sottoposti a test cutanei per aero-allergeni comuni, rinoscopia anteriore, rinomanometria anteriore attiva, fibrolaringoscopia e polisonnografia. Per la ricerca dei biomarkers dello stress ossidativo sono stati effettuati test diagnostici in campioni di sangue e urine. I gruppi sono risultati omogenei per età, sesso e distribuzione del Body Mass Index (BMI) (p0.05). Ci sono state differenze significative nell’AHI tra i tre gruppi di pazienti (p0.05). Nessuna significatività statistica è stata identificata (p0.05) tra i livelli di biomarkers di stress ossidativo nelle tre popolazioni studiate. I risultati del nostro studio hanno mostrato che il naso può svolgere un ruolo nella patogenesi dell’ OSAS, attraverso la produzione di biomarkers di stress ossidativo.

Lutein may have important antioxidant actions in free-radical-mediated diseases, in addition to its well-known antioxidant and cytoprotective effects on macula and photoreceptors. The peculiar perinatal susceptibility to oxidative stress indicates that prophylactic use of antioxidants as lutein could help to prevent or at least to reduce oxidative stress related diseases in newborns. Since lutein is not synthesized by humans, the intake primarily depends on diet or supplementation. Newborns receive lutein exclusively from breast milk. Lutein supplementation in term newborns has been reported to reduce oxidative stress and increase antioxidant capacities in the first days of life. Innovative frontiers concerning lutein supplementation are orientated toward cardiometabolic health improvement and cognitive benefits. The safety of lutein as an antioxidant agent has been confirmed in experimental and clinical studies, but its routine use is not recommended in perinatal period. This review summarizes what is known about the role of lutein as an antioxidant and anti-inflammatory agent in animal model and humans.

Maternal infection/inflammation represents one of the most important factors involved in the etiology of brain injury in newborns. We investigated the modulating effect of prenatal melatonin on the neonatal brain inflammation process resulting from maternal intraperitoneal (i.p.) lipopolysaccharide (LPS) injections. LPS (300 μg/kg) was administered to pregnant rats at gestational days 19 and 20. Melatonin (5 mg/kg) was administered i.p. at the same time as LPS. Melatonin counteracted the LPS sensitization to a second ibotenate-induced excitotoxic insult performed on postnatal day (PND) 4. As melatonin succeeded in reducing microglial activation in neonatal brain at PND1, pathways previously implicated in brain inflammation regulation, such as endoplasmic reticulum (ER) stress, autophagy and silent information regulator 1 (SIRT1), a melatonin target, were assessed at the same time-point in our experimental groups. Results showed that maternal LPS administrations resulted in an increase in CHOP and Hsp70 protein expression and eIF2α phosphorylation, indicative of activation of the unfolded protein response consequent to ER stress, and a slighter decrease in the autophagy process, determined by reduced lipidated LC3 and increased p62 expression. LPS-induced inflammation also reduced brain SIRT1 expression and affected the expression of miR-34a, miR146a, and miR-126. All these effects were blocked by melatonin. Cleaved-caspase-3 apoptosis pathway did not seem to be implicated in the noxious effect of LPS on the PND1 brain. We conclude that melatonin is effective in reducing maternal LPS-induced neonatal inflammation and related brain injury. Its role as a prophylactic/therapeutic drug deserves to be investigated by clinical studies.

Background Prenatal conditions of enhanced oxidative stress (OS) linked to inflammation or hypoxia have been associated with impaired fetal growth and preterm delivery. Little is known regarding biomarkers of OS in the cord blood of preterm infants and placental histological patterns. Objectives To test the hypothesis that placental lesions indicating chorioamnionitis (CA) or vascular underperfusion (VU) are associated with increased OS in the offspring. Methods 120 neonates born below 29+6 weeks of gestational age (GA) were enrolled. Histological characteristics of placentas from their mothers were classified as normal (CTRL group), histological CA (HCA) and vascular underperfusion (VU). Serum concentrations of isoprostanes (IsoPs), non-protein bound iron (NPBI) and advanced oxidative protein products (AOPP), were determined in cord blood. Results IsoPs, NPBI and AOPP were significantly increased in HCA group compared to CTRL group. The multivariable regression model, adjusted for GA, maternal age, parity, maternal diabetes, maternal obesity and presence/absence of fetal growth restriction (FGR), showed a significant association between the presence of HCA and increased OS biomarkers levels in cord blood (IsoPs: p = 0.006; NPBI: p = 0.014; AOPP: p = 0.007). Placental VU lesions were significantly associated with higher umbilical IsoPs, NPBI and AOPP levels (IsoPs: p = 0.008; NPBI: p = 0.002; AOPP: p = 0.040). In the cases of placental VU lesions associations were also found between high AOPP levels and low GA (p = 0.002) and the presence of fetal growth restriction (p = 0.014). Conclusions Placental lesions indicating inflammation or impaired perfusion are associated with higher cord blood levels of OS biomarkers explaining the fetal susceptibility to oxidative injury and the need of antioxidant protection.

Oxidative stress is a distinctive sign in several genetic disorders characterized by cancer predisposition, such as Ataxia-Telangiectasia, Fanconi Anemia, Down syndrome, progeroid syndromes, Beckwith-Wiedemann syndrome, and Costello syndrome. Recent literature unveiled new molecular mechanisms linking oxidative stress to the pathogenesis of these conditions, with particular regard to mitochondrial dysfunction. Since mitochondria are one of the major sites of ROS production as well as one of the major targets of their action, this dysfunction is thought to be the cause of the prooxidant status. Deeper insight of the pathogenesis of the syndromes raises the possibility to identify new possible therapeutic targets. In particular, the use of mitochondrial-targeted agents seems to be an appropriate clinical strategy in order to improve the quality of life and the life span of the patients.

Oxidative stress (OS) plays a key role in perinatal brain damage. The aim of this study is to evaluate the effectiveness of melatonin as a neuroprotective drug by investigating the influence of melatonin on OS and inflammation biomarkers in an animal model of cerebral hypoxia-ischemia.Five minutes after hypoxic-ischemic (HI) injury melatonin was administered to 28 rats (HI-Mel group). At the same time, 28 hypoxic-ischemic rats were vehicle-treated (V-HI group). Five rats were used as sham operated controls (CTL). OS biomarkers: isoprostanes (IsoPs), neuroprostanes (NPs) and neurofurans (NFs), and microglial activation markers (glial fibrillary acidic protein [GFAP] and monoclonal antirat CD68 [ED1]) were measured in the cerebral cortex of the two lobes.A significant increase of IsoPs on the left lobe was observed in V-HI after 1 hour (h) from HI injury (p0.001); a significant increase of NPs on both side (p0.05) and a significant increase of NFs on the left (p0.05) were also observed in V-HI after 24 h. A significant increase of IsoPs on the left (p0.05) and of NPs on both lobes (p0.05) were observed in HI-Mel after 48 h. The ED1 and GFAP expression was lower in the HI-Mel brain tissue.Melatonin reduces OS and inflammatory cells recruitment and glial cells activation in cerebral cortex after neonatal HI damage. These results lay the groundwork for future clinical studies in infants.

Rapamycin, a lipophilic macrolide antibiotic, has been found to reduce injury in different models of neurodegenerative disorders. We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI) the neuroprotective effect of rapamycin was associated with increased autophagy and decreased caspase-3 activation. We show here that the strong reduction of caspase-3 activation after rapamycin was due, at least in part, to its effect on the intrinsic apoptotic mitochondrial pathway because after rapamycin treatment there was a marked reduction of Bax and Bad translocation to mitochondria, cytochrome c release, and caspase-3 activation. Poly (ADP-ribose) polymerase 1 (PARP-1) cleavage and the number of terminal dUDP nick-end labeling (TUNEL)-positive cells were also reduced. To assess how the antiapoptotic effect of rapamycin was linked to the strong autophagy signal induced by the drug, we blocked the formation of autophagosomes with 3-methyladenine (3MA). 3MA administered 10 min after rapamycin, elicited again Bax and Bad translocation to the mitochondria but did not cause cytochrome c release and caspase-3 activation. After 3MA treatment, cells underwent necrotic cell death. These data indicate that rapamycin administered before HI prevents the apoptotic signaling taking place through the mitochondrial pathway. We hypothesize that rapamycin confers a preconditioning-like protection and suggest that caution is necessary before using pharmacological agents targeting autophagy in neuroprotection because they could interfere with endogenous protective mechanisms.

Patent ductus arteriosus (PDA) is the most common cardiovascular abnormality of the preterm infant usually treated with ibuprofen (IBU). PDA is strictly related to oxidative stress (OS) in neonates. This study tests the hypothesis that OS occurs in neonates with PDA and that IBU treatment reduces OS. Forty-three preterm babies with gestational age (GA)

We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI) rapamycin administration increases autophagy, decreases apoptosis and significantly reduces brain damage. After HI, when autophagy is blocked neuronal cells rapidly progress toward necrotic cell death. The present study was undertaken to assess the potential role of activation of autophagic and phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathways in the neuroprotective effect of rapamycin. Rapamycin administration caused a significant reduction of 70 kDa S6 kinase (p70S6K) phosphorylation and a significant increase of the autophagic proteins Beclin 1 and microtubule-associated protein 1 light chain 3 (LC3), as of monodansylcadaverine (MDC) labeling in the lesioned side. The phosphorylation of Akt and cAMP response element binding protein (CREB) was increased in neuronal cells, and both p-Akt and p-CREB colocalized with Beclin 1. Wortmannin (WM) administration significantly reduced Akt and CREB phosphorylation as well as the neuroprotective effect of rapamycin but did not affect the phosphorylation of p70S6K, the expression of Beclin 1 and LC3, and MDC labeling. In contrast, 3-methyladenine (3MA) reduced the increased Beclin 1 expression, the MDC labeling and the neuroprotective effect of rapamycin without affecting Akt phosphorylation. However, both compounds significantly increased necrotic cell death. Taken together, these data indicate that in neonatal HI autophagy can be part of an integrated prosurvival signaling which includes the PI3K-Akt-mammalian target of rapamycin (mTOR) axis. When the autophagic or the PI3K-Akt-mTOR pathways are interrupted cells undergo necrotic cell death.

Preterm infants have diminished antioxidant defenses. Glutathione (GSH), the main intracellular antioxidant, increases upon amino acid (AA) administration in preterm infants, without an accompanying rise of the fractional synthesis rate of GSH (FSRGSH) This study investigated the mechanism behind this increased GSH concentration by determining GSH synthesis in the first days after birth using stable isotope techniques in very low-birth-weight (VLBW) infants receiving i.v. AAs. Advanced oxidized protein products (AOPPs) were determined to quantify oxidative stress. Eighteen infants (birth weight 989 +/- 241 g, gestational age of 27/7 +/- 1/7 weeks) were studied either on postnatal day 1 or 2 (7 or 31 h postnatally, respectively). Concentration of GSH increased with postnatal age (1.45 +/- 0.48 mM versus 1.99 +/- 0.40 mM, p = 0.019). FSRGSH was not significantly different, but the absolute synthesis rate of GSH (ASRGSH) tended to be higher in the infants studied on day 2 [8.1 +/- 2.7 mg/(kg . d) versus 10.6 +/- 2.4 mg/(kg . d), p = 0.054]. AOPP concentrations were not different between groups. In conclusion, GSH concentration in VLBW infants increases significantly after birth. A concomitant increased synthesis rate was not found, suggesting that GSH consumption decreases upon AA administration.

Despite the role of reactive oxygen species in the development of respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in preterm infants, the anti-oxidant properties of commercial surfactants have never been studied. We measured the superoxide dismutase (SOD) and catalase (CAT) activity, the scavenger activity against hydrogen peroxide (H2O2), and its changes after the addition of SOD and CAT in four natural surfactants, namely Infasurf, Curosurf, Survanta, and Alveofact. We found that they contain measurable amount of SOD and CAT. Curosurf and Survanta seem to have higher antioxidant effect than Infasurf and Alveofact. Moreover, the highest phospholipid concentration and recommended dose of Curosurf imply that its scavenger activity for each treatment dose in preterm infants is likely higher than that of Survanta. Finally, the supplementation with SOD and CAT induced a remarkable increase of antioxidant action in all studied surfactants. Pediatr Pulmonol. 2009; 44:1125–1131. ©2009 Wiley-Liss, Inc.