Fabrizio Proietti

Melatonin protects from the long-term consequences of a neonatal hypoxic-ischemic brain injury in rats

Abstract:  Among the main factors responsible for perinatal brain injury, inflammation, hypoxia‐ischemia and formation of free radicals (FR) appear to play key roles. Melatonin, an endogenously produced indoleamine formed in higher amounts in adults than in neonates, is a potent FR scavenger as well as an indirect antioxidant. Herein, we examined whether melatonin provides significant protection against brain damage and its long‐term consequences in a neonatal model of hypoxia‐ischemia (HI). Seven day‐old rats were subjected to permanent legation of the right common carotid artery followed to 2.5 hrs hypoxia 3 hrs later (HI). The neuroprotective effect of melatonin was evaluated 7 days after HI, or when rats reached adulthood, using behavioral and histological analyses. A beneficial effect was observed with 5 mg/kg melatonin administered before HI. The same dose repeated three times reduced further injury. A significant protective effect was found when 15 mg/kg melatonin was given 30 min before HI or when the same dose was given after HI and administration repeated after 24 and 48 hrs. The latter schedule of administration was used to assess the long‐term protective effects. Melatonin did not affect growth rate and behavior at adulthood, but significantly improved the behavioral asymmetry and learning deficits induced by HI. Consistently, brain injury was significantly attenuated in the melatonin‐treated ischemic group. The present study demonstrates that melatonin administration before or after HI in immature rats has an excellent and long‐lasting benefit on ischemic outcomes suggesting that the drug could represent a potentially safe approach to perinatal brain damage in humans.

Melatonin reduces endoplasmic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxia-ischemia

Conditions that interfere with the endoplasmic reticulum (ER) functions cause accumulation of unfolded proteins in the ER lumen, referred to as ER stress, and activate a homeostatic signaling network known as unfolded protein response (UPR). We have previously shown that in neonatal rats subjected to hypoxia–ischemia (HI), melatonin administration significantly reduces brain damage. This study assessed whether attenuation of ER stress is involved in the neuroprotective effect of melatonin after neonatal HI. We found that the UPR was strongly activated after HI. Melatonin significantly reduced the neuron splicing of XBP‐1 mRNA, the increased phosphorylation of eIF2α, and elevated expression of chaperone proteins GRP78 and Hsp70 observed after HI in the brain. CHOP, which plays a convergent role in the UPR, was reduced as well. Melatonin also completely prevented the depletion of SIRT‐1 induced by HI, and this effect was observed in the same neurons that over‐express CHOP. These results demonstrate that melatonin reduces ER stress induced by neonatal HI and preserves SIRT‐1 expression, suggesting that SIRT‐1, due to its action in the modulation of a wide variety of signaling pathways involved in neuroprotection, may play a key role in the reduction of ER stress and neuroprotection observed after melatonin.

Resuscitation with supplementary oxygen induces oxidative injury in the cerebral cortex

Isoprostanes, neuroprostanes, isofurans, and neurofurans have all become attractive biomarkers of oxidative damage and lipid peroxidation in brain tissue. Asphyxia and subsequent reoxygenation cause a burst of oxygen free radicals. Isoprostanes and isofurans are generated by free radical attacks of esterified arachidonic acid. Neuroprostanes and neurofurans are derived from the peroxidation of docosahexanoic acid, which is abundant in neurons and could therefore more selectively represent oxidative brain injury. Newborn piglets (age 12-36 h) underwent hypoxia until the base excess reached -20 mmol/L or the mean arterial blood pressure dropped below 15 mm Hg. They were randomly assigned to receive resuscitation with 21, 40, or 100% oxygen for 30 min and then ventilation with air. The levels of isoprostanes, isofurans, neuroprostanes, and neurofurans were determined in brain tissue (ng/g) isolated from the prefrontal cortex using gas chromatography-mass spectrometry (GC/MS) with negative ion chemical ionization (NICI) techniques. A control group underwent the same procedures and observations but was not submitted to hypoxia or hyperoxia. Hypoxia and reoxygenation significantly increased the levels of isoprostanes, isofurans, neuroprostanes, and neurofurans in the cerebral cortex. Nine hours after resuscitation with 100% oxygen for 30 min, there was nearly a 4-fold increase in the levels of isoprostanes and isofurans compared to the control group (P=0.007 and P=0.001) and more than a 2-fold increase in neuroprostane levels (P=0.002). The levels of neuroprostanes and neurofurans were significantly higher in the piglets that were resuscitated with supplementary oxygen (40 and 100%) compared to the group treated with air (21%). The significance levels of the observed differences in neuroprostanes for the 21% vs 40% comparison and the 21% vs 100% comparison were P<0.001 and P=0.001, respectively. For neurofurans, the P values of the 21% vs 40% comparison and the 21% vs 100% comparison were P=0.036 and P=0.025, respectively. Supplementary oxygen used for the resuscitation of newborns increases lipid peroxidation in brain cortical neurons, a result that is indicative of oxidative brain damage. These novel findings provide new knowledge regarding the relationships between oxidative brain injury and resuscitation with oxygen.

The use of melatonin in hypoxic-ischemic brain damage: an experimental study

Objective: 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. Methods: 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. Results: A significant increase of IsoPs on the left lobe was observed in V-HI after 1 hour (h) from HI injury (p < 0.001); a significant increase of NPs on both side (p < 0.05) and a significant increase of NFs on the left (p < 0.05) were also observed in V-HI after 24 h. A significant increase of IsoPs on the left (p < 0.05) and of NPs on both lobes (p < 0.05) were observed in HI-Mel after 48 h. The ED1 and GFAP expression was lower in the HI-Mel brain tissue. Conclusions: 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.

Increased autophagy reduces endoplasmic reticulum stress after neonatal hypoxia–ischemia: Role of protein synthesis and autophagic pathways

The endoplasmic reticulum (ER) stress can result from several pathological conditions that perturb ER homeostasis and is characterized by accumulation of unfolded proteins in the ER lumen. To cope with ER stress, cells activate the unfolded protein response (UPR), a protein quality control mechanism aimed at restoring homeostasis. The present study was undertaken to characterize the UPR after neonatal hypoxia/ischemia (HI) and its crosstalk with autophagy. After HI, there was a significant increase of GRP78 and Hsp70 expression, phosphorylation of eIF2α, Xbp-1 mRNA splicing and CHOP expression, revealing severe ER stress and UPR. Increasing autophagy with rapamycin (Rap) significantly reduced the UPR. Rap did not further increase the eIF2α phosphorylation and p70S6 kinase (p70S6K) inactivation induced by HI. After autophagy activation, however, there was a clear co-localization between monodansylcadaverine (MDC)-positive autophagosome-like structures and the ribosomal protein S6 (RPS6), indicating the presence of ribosomes in autophagosomes (ribophagy). We found that the autophagy inhibitor 3-methyladenine administered after Rap treatment completely reverted the increased phosphorylation of eIF2α and p70S6K inactivation, and blocked the formation of autophagosome-like structures restoring the UPR. These results demonstrate that the UPR is strongly activated after neonatal HI. Over-activation of autophagy significantly reduces this response, highlighting the relevance of the cross-talk between ER and the autophagy machinery in this important pathological condition. Furthermore, the presence of ribosome subunits in autophagosome-like structures suggests that increased ribosome turnover through autophagy (ribophagy) may represent an additional mechanism involved in the neuroprotective effect observed after autophagy over-activation.

May oxidative stress biomarkers in cord blood predict the occurrence of necrotizing enterocolitis in preterm infants

Introduction: Oxidative stress (OS) is strongly involved in the pathogenesis of many preterm newborn diseases; this is due to the low efficiency of neonatal antioxidant systems unable to counteract the harmful effects of free radicals (FRs). Hypoxic-ischemic events and inflammation, involved in necrotizing enterocolitis (NEC) pathogenesis, are responsible of the overproduction of FRs, generating OS. Aim: To test the hypotesis that OS markers levels in cord blood may early identify the newborns at high risk to develop NEC. Materials and methods: 332 preterm newborns of gestational age (GA) between 24 and 33 week and birth weight (BW) between 460 and 2540 g were consecutively recruited in three european neonatal intensive care units. Markers of potential OS risk: non-protein bound iron (NPBI), and markers of FRs damage: advanced oxidation protein products (AOPP) and total hydroperoxides (TH), were measured in the cord blood. Associations between NEC and OS markers were checked through inferential analysis. Results: Out of 332 preterm babies, 29 developed NEC. Babies with NEC had a BW and a GA significantly lower than healthy babies. AOPP, TH and NPBI cord blood levels were significantly higher in babies with NEC than in babies without (respectively mean AOPP = 28.05 ± 21 vs 15.80 ± 7.14; p < 0.05; TH = 154.48 ± 84.67 vs 107.40 ± 61.01; p < 0.05; NPBI = 2.21 ± 3.98 vs 0.95 ± 1.59; p < 0.05). Conclusions: The determination of OS biomarkers in cord blood can be useful in identifying babies at high risk for NEC and in devising new strategies to ameliorate perinatal outcome.

Effects of Lutein on Oxidative Stress in the Term Newborn: A Pilot Study

Background: Oxidative stress (OS) plays a crucial role in pathological conditions during the early neonatal period. The newborns are susceptible to oxidative damage due to high metabolic rate and low levels of antioxidant enzymes. Lutein has been found to have protective functions in adult humans as antioxidant. Aim: To evaluate the effects of lutein on OS in newborns. We tested the hypothesis that lutein would act both by increasing antioxidant capacity and inhibiting OS. Methods: This was a randomized, double-blind, placebo-controlled, single-center study. 20 healthy term newborns were assigned to receive lutein or placebo (lutein and control group, respectively) at 12 and 36 h after birth. Total hydroperoxides (TH), as marker of OS, and biological antioxidant potential (BAP), as marker of antioxidant power, were detected on cord blood and at 48 h of life in all babies. Results: TH significantly increased from birth to 48 h in the control group (p = 0.02), but not in the lutein group. In the lutein group, BAP significantly increased after 48 h (p = 0.02), showing a strengthening of antioxidant activity due to lutein. At 48 h of life, compared with those in the control group, neonates assigned to receive lutein had significantly lower TH levels (p = 0.04) and higher BAP levels (p = 0.028). Conclusions: Lutein administration in newborns increases the levels of BAP decreasing TH. The enhancement of antioxidant activity in plasma clearly results in protecting newborn from perinatal OS. These preliminary results, adding a new contribution in antioxidant strategies, strongly require to be confirmed by RCT.

Inhibition of rapamycin-induced autophagy causes necrotic cell death associated with Bax/Bad mitochondrial translocation

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.

Lipid and Protein Oxidation in Newborn Infants after Lutein Administration

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.gov NCT02068807.

Oxidative stress in children affected by epileptic encephalopathies.

Oxidative stress may lead to abnormal peroxidation of membrane lipids, oxidation of sulfhydryl groups and disruption of nucleic acids. Experimental and clinical studies suggested that free radicals may be involved in the pathogenesis of epilepsy. Three groups of patients were considered in the study. Group 1 (N=34) included patients affected by epileptic encephalopathy; Group 2 (N=31) included those affected by idiopathic epilepsy syndromes and under valproic acid (VPA) monotherapy, and Group 3 (N=22) represented by healthy controls. All patients and healthy children underwent blood withdrawals to evaluate redox status by measuring levels of F2-isoprostanes (F2-iso), advanced oxidative protein products (AOPP), non-protein binding iron (NPBI), thiols (-SH groups), and total hydroperoxides (TH). In comparison to the controls, Group 1 patients showed significantly higher plasma levels of F2-iso, AOPP, and TH. By contrast, no differences there were in the plasma NPBI concentrations. Again, no statistical differences there were in the plasma levels of the oxidative stress markers between patients from Group 2 and normal subjects. Our study showed that patients with epileptic encephalopathy have increased levels of oxidative stress markers. By contrast, normal redox status was observed in patients with idiopathic epilepsy syndromes under long-term VPA monotherapy.