Neuro-Specific and Immuno-Inflammatory Biomarkers in Umbilical Cord Blood in Neonatal Hypoxic-Ischemic Encephalopathy.

OBJECTIVES: The aim of the study was to evaluate neuronal injury and immuno-inflammatory biomarkers in umbilical cord blood (UCB) at birth, in cases with perinatal asphyxia with or without hypoxic-ischemic encephalopathy (HIE), compared with healthy controls and to assess their ability to predict HIE. STUDY DESIGN: In this case-control study, term infants with perinatal asphyxia were recruited at birth. UCB was stored at delivery for batch analysis. HIE was diagnosed by clinical Sarnat staging at 24 h. Glial fibrillary acidic protein (GFAP), the neuronal biomarkers tau and neurofilament light protein (NFL), and a panel of cytokines were analyzed in a total of 150 term neonates: 50 with HIE, 50 with asphyxia without HIE (PA), and 50 controls. GFAP, tau, and NFL concentrations were measured using ultrasensitive single-molecule array (Simoa) assays, and a cytokine screening panel was applied to analyze the immuno-inflammatory and infectious markers. RESULTS: GFAP, tau, NFL, and several cytokines were significantly higher in newborns with moderate and severe HIE compared to a control group and provided moderate prediction of HIE II/III (AUC: 0.681-0.827). Furthermore, the levels of GFAP, tau, interleukin-6 (IL-6), and interleukin-8 (IL-8) were higher in HIE II/III cases compared with cases with PA/HIE I. IL-6 was also higher in HIE II/III compared with HIE I cases. CONCLUSIONS: Biomarkers of brain injury and inflammation were increased in umbilical blood in cases with asphyxia. Several biomarkers were higher in HIE II/III versus those with no HIE or HIE I, suggesting that they could assist in the prediction of HIE II/III.

Noninvasive monitoring of evolving urinary metabolic patterns in neonatal encephalopathy.

BACKGROUND: Infants with moderate and severe neonatal encephalopathy (NE) frequently suffer from long-term adverse outcomes. We hypothesize that the urinary metabolome of newborns with NE reflects the evolution of injury patterns observed with magnetic resonance imaging (MRI). METHODS: Eligible patients were newborn infants with perinatal asphyxia evolving to NE and qualifying for therapeutic hypothermia (TH) included in the HYPOTOP trial. MRI was employed for characterizing brain injury. Urine samples of 55 infants were collected before, during, and after TH. Metabolic profiles of samples were recorded employing three complementary mass spectrometry-based assays, and the alteration of detected metabolic features between groups was assessed. RESULTS: The longitudinal assessment revealed significant perturbations of the urinary metabolome. After 24 h of TH, a stable disease pattern evolved characterized by the alterations of 4-8% of metabolic features related to lipid metabolism, metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, amino acid metabolism, and nucleotide metabolism. Characteristic metabolomic fingerprints were observed for different MRI injury patterns. CONCLUSIONS: This study shows the potential of urinary metabolic profiles for the noninvasive monitoring of brain injury of infants with NE during TH. IMPACT: A comprehensive approach for the study of the urinary metabolome was employed involving a semi-targeted capillary electrophoresis-time-of-flight mass spectrometry (TOFMS) assay, an untargeted ultra-performance liquid chromatography (UPLC)-quadrupole TOFMS assay, and a targeted UPLC-tandem MS-based method for the quantification of amino acids. The longitudinal study of the urinary metabolome identified dynamic metabolic changes between birth and until 96 h after the initiation of TH. The identification of altered metabolic pathways in newborns with pathologic MRI outcomes might offer the possibility of developing noninvasive monitoring approaches for personalized adjustment of the treatment and for supporting early outcome prediction.

Mitochondrial dysfunction in perinatal asphyxia: role in pathogenesis and potential therapeutic interventions.

Perinatal asphyxia (PA)-induced brain injury may present as hypoxic-ischemic encephalopathy in the neonatal period, and long-term sequelae such as spastic motor deficits, intellectual disability, seizure disorders and learning disabilities. The brain injury is secondary to both the hypoxic-ischemic event and oxygenation-reperfusion following resuscitation. Following PA, a time-dependent progression of neuronal insult takes place in terms of transition of cell death from necrosis to apoptosis. This transition is the result of time-dependent progression of pathomechanisms which involve excitotoxicity, oxidative stress, and ultimately mitochondrial dysfunction in developing brain. More precisely mitochondrial respiration is suppressed and calcium signalling is dysregulated. Consequently, Bax-dependent mitochondrial permeabilization occurs leading to release of cytochrome c and activation of caspases leading to transition of cell death in developing brain. The therapeutic window lies within this transition process. At present, therapeutic hypothermia (TH) is the only clinical treatment available for treating moderate as well as severe asphyxia in new-born as it attenuates secondary loss of high-energy phosphates (ATP) (Solevåg et al. in Free Radic Biol Med 142:113-122, 2019; Gunn et al. in Pediatr Res 81:202-209, 2017), improving both short- and long-term outcomes. Mitoprotective therapies can offer a new avenue of intervention alone or in combination with therapeutic hypothermia for babies with birth asphyxia. This review will explore these mitochondrial pathways, and finally will summarize past and current efforts in targeting these pathways after PA, as a means of identifying new avenues of therapeutic intervention.

The Kynurenic Acid Analog SZR72 Enhances Neuronal Activity after Asphyxia but Is Not Neuroprotective in a Translational Model of Neonatal Hypoxic Ischemic Encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) remains to be a major cause of long-term neurodevelopmental deficits in term neonates. Hypothermia offers partial neuroprotection warranting research for additional therapies. Kynurenic acid (KYNA), an endogenous product of tryptophan metabolism, was previously shown to be beneficial in rat HIE models. We sought to determine if the KYNA analog SZR72 would afford neuroprotection in piglets. After severe asphyxia (pHa = 6.83 ± 0.02, ΔBE = -17.6 ± 1.2 mmol/L, mean ± SEM), anesthetized piglets were assigned to vehicle-treated (VEH), SZR72-treated (SZR72), or hypothermia-treated (HT) groups (n = 6, 6, 6; Tcore = 38.5, 38.5, 33.5 °C, respectively). Compared to VEH, serum KYNA levels were elevated, recovery of EEG was faster, and EEG power spectral density values were higher at 24 h in the SZR72 group. However, instantaneous entropy indicating EEG signal complexity, depression of the visual evoked potential (VEP), and the significant neuronal damage observed in the neocortex, the putamen, and the CA1 hippocampal field were similar in these groups. In the caudate nucleus and the CA3 hippocampal field, neuronal damage was even more severe in the SZR72 group. The HT group showed the best preservation of EEG complexity, VEP, and neuronal integrity in all examined brain regions. In summary, SZR72 appears to enhance neuronal activity after asphyxia but does not ameliorate early neuronal damage in this HIE model.

Effects of therapeutic hypothermia on death among asphyxiated neonates with hypoxic-ischemic encephalopathy: A systematic review and meta-analysis of randomized control trials.

BACKGROUND: Hypoxic perinatal brain injury is caused by lack of oxygen to baby's brain and can lead to death or permanent brain damage. However, the effectiveness of therapeutic hypothermia in birth asphyxiated infants with encephalopathy is uncertain. This systematic review and meta-analysis was aimed to estimate the pooled relative risk of mortality among birth asphyxiated neonates with hypoxic-ischemic encephalopathy in a global context. METHODS: We used the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines to search randomized control trials from electronic databases (PubMed, Cochrane library, Google Scholar, MEDLINE, Embase, Scopus, Web of Science, Cochrane Central Register of Controlled Trials (CENTRAL), and meta register of Current Controlled Trials (mCRT)). The authors extracted the author's name, year of publication, country, method of cooling, the severity of encephalopathy, the sample size in the hypothermic, and non-hypothermic groups, and the number of deaths in the intervention and control groups. A weighted inverse variance fixed-effects model was used to estimate the pooled relative risk of mortality. The subgroup analysis was done by economic classification of countries, methods of cooling, and cooling devices. Publication bias was assessed with a funnel plot and Eggers test. A sensitivity analysis was also done. RESULTS: A total of 28 randomized control trials with a total sample of 35, 92 (1832 hypothermic 1760 non-hypothermic) patients with hypoxic-ischemic encephalopathy were used for the analysis. The pooled relative risk of mortality after implementation of therapeutic hypothermia was found to be 0.74 (95%CI; 0.67, 0.80; I2 = 0.0%; p<0.996). The subgroup analysis revealed that the pooled relative risk of mortality in low, low middle, upper-middle and high income countries was 0.32 (95%CI; -0.95, 1.60; I2 = 0.0%; p<0.813), 0.5 (95%CI; 0.14, 0.86; I2 = 0.0%; p<0.998), 0.62 (95%CI; 0.41-0.83; I2 = 0.0%; p<0.634) and 0.76 (95%CI; 0.69-0.83; I2 = 0.0%; p<0.975) respectively. The relative risk of mortality was the same in selective head cooling and whole-body cooling method which was 0.74. Regarding the cooling device, the pooled relative risk of mortality is the same between the cooling cap and cooling blanket (0.74). However, it is slightly lower (0.73) in a cold gel pack. CONCLUSIONS: Therapeutic hypothermia reduces the risk of death in neonates with moderate to severe hypoxic-ischemic encephalopathy. Both selective head cooling and whole-body cooling method are effective in reducing the mortality of infants with this condition. Moreover, low income countries benefit the most from the therapy. Therefore, health professionals should consider offering therapeutic hypothermia as part of routine clinical care to newborns with hypoxic-ischemic encephalopathy especially in low-income countries.

Aberrant expression of miR-199a in newborns with hypoxic-ischemic encephalopathy and its diagnostic and prognostic significance when combined with S100B and NSE.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a disorder mainly due to asphyxia during the perinatal period, and late diagnosis leads to high mortality. In this study, the expression of microRNA-199a (miR-199a) in HIE newborns was investigated, as well as its clinical significance in HIE diagnosis and prognosis. Circulating levels of S100B and NSE in HIE newborns were measured using enzyme-linked immunosorbent assay, and the expression of miR-199a was analyzed using quantitative real-time PCR. The diagnostic value of miR-199a, S100B and NSE was evaluated using the receiver operating characteristic (ROC) analysis, and their prognostic value was assessed by the evaluation of Gesell intellectual development of the HIE newborns. HIE newborns possessed significantly increased levels of S100B and NSE and decreased miR-199a (all P < 0.01). The Neonatal Behavioral Neurological Assessment (NBNA) score of HIE newborns was negatively correlated with S100B and NSE, while was positively correlated miR-199a. The ROC analysis results showed the diagnostic value of serum miR-199a, and the combined detection of miR-199a, S100B and NSE could obtained the highest diagnostic accuracy in HIE newborns. miR-199a expression was lowest in newborns with severe HIE, and it had diagnostic potential to distinguish HIE cases with different severity. Regarding the prognosis of neonatal HIE, the correlation of miR-199a, S100B, NSE with Gesell intellectual development was found in HIE newborns. The decreased miR-199a in HIE newborns serves as a potential diagnostic biomarker and may help to improve the diagnostic and prognostic value of S100B and NSE in neonatal HIE.

Excess cerebral oxygen delivery follows return of spontaneous circulation in near-term asphyxiated lambs.

Hypoxic-ischaemia renders the neonatal brain susceptible to early secondary injury from oxidative stress and impaired autoregulation. We aimed to describe cerebral oxygen kinetics and haemodynamics immediately following return of spontaneous circulation (ROSC) and evaluate non-invasive parameters to facilitate bedside monitoring. Near-term sheep fetuses [139 ± 2 (SD) days gestation, n = 16] were instrumented to measure carotid artery (CA) flow, pressure, right brachial arterial and jugular venous saturation (SaO2 and SvO2, respectively). Cerebral oxygenation (crSO2) was measured using near-infrared spectroscopy (NIRS). Following induction of severe asphyxia, lambs received cardiopulmonary resuscitation using 100% oxygen until ROSC, with oxygen subsequently weaned according to saturation nomograms as per current guidelines. We found that oxygen consumption did not rise following ROSC, but oxygen delivery was markedly elevated until 15 min after ROSC. CrSO2 and heart rate each correlated with oxygen delivery. SaO2 remained > 90% and was less useful for identifying trends in oxygen delivery. CrSO2 correlated inversely with cerebral fractional oxygen extraction. In conclusion, ROSC from perinatal asphyxia is characterised by excess oxygen delivery that is driven by rapid increases in cerebrovascular pressure, flow, and oxygen saturation, and may be monitored non-invasively. Further work to describe and limit injury mediated by oxygen toxicity following ROSC is warranted.

Animal models for neonatal brain injury induced by hypoxic ischemic conditions in rodents.

Neonatal hypoxia-ischemia and resulting encephalopathies are of significant concern. Intrapartum asphyxia is a leading cause of neonatal death globally. Among surviving infants, there remains a high incidence of hypoxic-ischemic encephalopathy due to neonatal hypoxic-ischemic brain injury, manifesting as mild conditions including attention deficit hyperactivity disorder, and debilitating disorders such as cerebral palsy. Various animal models of neonatal hypoxic brain injury have been implemented to explore cellular and molecular mechanisms, assess the potential of novel therapeutic strategies, and characterize the functional and behavioural correlates of injury. Each of the animal models has individual advantages and limitations. The present review looks at several widely-used and alternative rodent models of neonatal hypoxia and hypoxia-ischemia; it highlights their strengths and limitations, and their potential for continued and improved use.

Brain interstitial pH changes in the subacute phase of hypoxic-ischemic encephalopathy in newborn pigs.

Brain interstitial pH (pHbrain) alterations play an important role in the mechanisms of neuronal injury in neonatal hypoxic-ischemic encephalopathy (HIE) induced by perinatal asphyxia. The newborn pig is an established large animal model to study HIE, however, only limited information on pHbrain alterations is available in this species and it is restricted to experimental perinatal asphyxia (PA) and the immediate reventilation. Therefore, we sought to determine pHbrain over the first 24h of HIE development in piglets. Anaesthetized, ventilated newborn pigs (n = 16) were instrumented to control major physiological parameters. pHbrain was determined in the parietal cortex using a pH-selective microelectrode. PA was induced by ventilation with a gas mixture containing 6%O2-20%CO2 for 20 min, followed by reventilation with air for 24h, then the brains were processed for histopathology assessment. The core temperature was maintained unchanged during PA (38.4±0.1 vs 38.3±0.1°C, at baseline versus the end of PA, respectively; mean±SEM). In the arterial blood, PA resulted in severe hypoxia (PaO2: 65±4 vs 23±1*mmHg, *p<0.05) as well as acidosis (pHa: 7.53±0.03 vs 6.79±0.02*) that is consistent with the observed hypercapnia (PaCO2: 37±3 vs 160±6*mmHg) and lactacidemia (1.6±0.3 vs 10.3±0.7*mmol/L). Meanwhile, pHbrain decreased progressively from 7.21±0.03 to 5.94±0.11*. Reventilation restored pHa, blood gases and metabolites within 4 hours except for PaCO2 that remained slightly elevated. pHbrain returned to 7.0 in 29.4±5.5 min and then recovered to its baseline level without showing secondary alterations during the 24 h observation period. Neuropathological assessment also confirmed neuronal injury. In conclusion, in spite of the severe acidosis and alterations in blood gases during experimental PA, pHbrain recovered rapidly and notably, there was no post-asphyxia hypocapnia that is commonly observed in many HIE babies. Thus, the neuronal injury in our piglet model is not associated with abnormal pHbrain or low PaCO2 over the first 24 h after PA.

The Effect of Size, Maturation, Global Asphyxia, Cerebral Ischemia, and Therapeutic Hypothermia on the Pharmacokinetics of High-Dose Recombinant Erythropoietin in Fetal Sheep.

High-dose human recombinant erythropoietin (rEPO) is a promising potential neuroprotective treatment in preterm and full-term neonates with hypoxic-ischemic encephalopathy (HIE). There are limited data on the pharmacokinetics of high-dose rEPO in neonates. We examined the effects of body weight, gestation age, global asphyxia, cerebral ischemia, hypothermia and exogenous rEPO on the pharmacokinetics of high-dose rEPO in fetal sheep. Near-term fetal sheep on gestation day 129 (0.87 gestation) (full term 147 days) received sham-ischemia (n = 5) or cerebral ischemia for 30 min followed by treatment with vehicle (n = 4), rEPO (n = 8) or combined treatment with rEPO and hypothermia (n = 8). Preterm fetal sheep on gestation day 104 (0.7 gestation) received sham-asphyxia (n = 1) or complete umbilical cord occlusion for 25 min followed by i.v. infusion of vehicle (n = 8) or rEPO (n = 27) treatment. rEPO was given as a loading bolus, followed by a prolonged continuous infusion for 66 to 71.5 h in preterm and near-term fetuses. A further group of preterm fetal sheep received repeated bolus injections of rEPO (n = 8). The plasma concentrations of rEPO were best described by a pharmacokinetic model that included first-order and mixed-order elimination with linear maturation of elimination with gestation age. There were no detectable effects of therapeutic hypothermia, cerebral ischemia, global asphyxia or exogenous treatment on rEPO pharmacokinetics. The increase in rEPO elimination with gestation age suggests that to maintain target exposure levels during prolonged treatment, the dose of rEPO may have to be adjusted to match the increase in size and growth. These results are important for designing and understanding future studies of neuroprotection with high-dose rEPO.

Rates of metabolic acidosis at birth and Apgar score values at 1, 5, and 10 min in term infants: a Swedish cohort study.

Background Metabolic acidosis, measured in arterial umbilical cord blood at birth, is the most accepted definition of birth asphyxia. The aim of the study was to investigate the rates of metabolic acidosis across the entire range of Apgar score values (0-10) at 1, 5, and 10 min in term infants. Methods In a population-based Swedish cohort of births between 2008 and 2013, we included 85,076 term (≥37 weeks) non-malformed infants with information from umbilical arterial blood gas analyses and complete information on Apgar scores (0-10) at 1, 5, and 10 min. Results Rates of metabolic acidosis generally decreased with increasing Apgar score values. For Apgar score at 1 min, this decrease was consistent from Apgar score 0 (35%) to Apgar score 10 (0%). For Apgar scores at 5 and 10 min, the decrease was consistent for Apgar score values from 6 to 10. Conclusion Although there is a close association between Apgar score values and rates of metabolic acidosis, Apgar score is not and should not be used as a measure of birth asphyxia.

Neonatal hypoxia-ischemia in rat elicits a region-specific neurotrophic response in SVZ microglia.

BACKGROUND: Recent findings describe microglia as modulators of neurogenesis in the subventricular zone (SVZ). SVZ microglia in the adult rat are thought to adopt a neurotrophic phenotype after ischemic stroke. Early postnatal microglia are endogenously activated and may therefore exhibit an increased sensitivity to neonatal hypoxia-ischemia (HI). The goal of this study was to investigate the impact of cortico-striatal HI on the microglial phenotype, function, and gene expression in the early postnatal SVZ. METHODS: Postnatal day (P)7 rats underwent sham or right-hemispheric HI surgery. Microglia in the SVZ, the uninjured cortex, and corpus callosum were immunohistochemically analyzed at P10, P20, and P40. The transcriptome of microdissected SVZ and cortical microglia was analyzed at P10 and P20, and the effect of P10 SVZ microglia on neurosphere generation in vitro was studied. RESULTS: The microglial response to HI was region-specific. In the SVZ, a microglial accumulation, prolonged activation and phagocytosis was noted that was not observed in the cortex and corpus callosum. The transcriptome of SVZ microglia and cortical microglia were distinct, and after HI, SVZ microglia concurrently upregulated pro- and anti-inflammatory as well as neurotrophic genes. In vitro, microglia isolated from the SVZ supported neurosphere generation in a concentration-dependent manner. CONCLUSIONS: Microglia are an inherent cellular component of the early postnatal SVZ and undergo developmental changes that are affected on many aspects by neonatal HI injury. Our results demonstrate that early postnatal SVZ microglia are sensitive to HI injury and display a long-lasting region-specific response including neurotrophic features.

Thioredoxin 1 Plays a Protective Role in Retinas Exposed to Perinatal Hypoxia-Ischemia.

Thioredoxin family proteins are key modulators of cellular redox regulation and have been linked to several physiological functions, including the cellular response to hypoxia-ischemia. During perinatal hypoxia-ischemia (PHI), the central nervous system is subjected to a fast decrease in O2 and nutrients with a subsequent reoxygenation that ultimately leads to the production of reactive species impairing physiological redox signaling. Particularly, the retina is one of the most affected tissues, due to its high oxygen consumption and exposure to light. One of the main consequences of PHI is retinopathy of prematurity, comprising changes in retinal neural and vascular development, with further compensatory mechanisms that can ultimately lead to retinal detachment and blindness. In this study, we have analyzed long-term changes that occur in the retina using two well established in vivo rat PHI models (perinatal asphyxia and carotid ligation model), as well as the ARPE-19 cell line that was exposed to hypoxia and reoxygenation. We observed significant changes in the protein levels of the cytosolic oxidoreductase thioredoxin 1 (Trx1) in both animal models and a cell model. Knock-down of Trx1 in ARPE-19 cells affected cell morphology, proliferation and the levels of specific differentiation markers. Administration of recombinant Trx1 decreased astrogliosis and improved delayed neurodevelopment in animals exposed to PHI. Taken together, our results suggest therapeutical implications for Trx1 in retinal damage induced by hypoxia-ischemia during birth.

Prospective qualification of early cerebral biomarkers in a randomised trial of treatment with xenon combined with moderate hypothermia after birth asphyxia.

BACKGROUND: The TOBY-Xe proof of concept randomised trial found no effect of xenon combined with hypothermia after birth asphyxia on the lactate to N-acetyl aspartate ratio (Lac/NAA) in the thalamus and fractional anisotropy (FA) in white matter tracts measured within 15 days of birth. To confirm that these biomarkers are qualified to predict long-term outcome after neural rescue therapy we assessed surviving participants at 2-3 years of age. METHODS: Of the 92 infants in TOBY-Xe, one was omitted from the study, 69 survived and we assessed 62 participants, 32 in the hypothermia and xenon and 30 in the hypothermia only groups. We examined the relation between Lac/NAA and FA and the scores of the Bayley Scales of Infant and Toddler Development III and calculated their predictive accuracy for moderate or severe disability or death. RESULTS: Fifteen of 62 participants (24%) developed moderate/severe disability, and 22/92 (24%) died. The Lac/NAA ratio (difference in medians 0.628, 95% CI -0.392 to 4.684) and FA (difference in means -0.055, 95% CI -0.033 to -0.077) differed significantly between participants with or without moderate or severe disability or death and were significantly related with development scores in both groups. Adverse outcomes were correctly identified in 95.65% of cases by Lac/NAA and 78.79% by FA, with adequate mean calibration of the model. INTERPRETATION: The results confirm the qualification of the cerebral magnetic resonance biomarkers employed in the TOBY-Xe study as predictors of outcome after neuroprotective therapy. FUND: The Centre for the Developing Brain, King's College London, UK.

Thermal conditions during neonatal anoxia affect the endogenous level of brain-derived neurotrophic factor.

Anoxia during delivery is a complication that can disturb infant brain development leading to various types of neurological disorders. Our studies have shown that increased body temperature of newborn rats of both sexes intensifies the postanoxic oxidative stress and prevents triggering the endogenous adaptive response such as HIF-1α activation. Currently, brain-derived neurotrophic factor-BDNF is considered to be a modulator of neuronal plasticity. In the developing brain, mature BDNF and its precursor exhibit prosurvival action through the TrkB receptor and proapoptotic functions binding to p75NTR , respectively. The aim of our experiments was to check the effects of body temperature on the postanoxic level of BDNF and on the expression of its receptors as well as on the marker of apoptosis-caspase-3 in the rat brain. Two-day-old Wistar Han rats (male/female ratio, 1:1) were exposed to anoxia in 100% nitrogen atmosphere for 10 min in different thermal conditions, which allowed them to regulate their rectal temperature at the following levels: normothermic-33°C; hyperthermic-37°C; and extremely hyperthermic-39°C. Thermal conditions during neonatal anoxia affected the level of proBDNF, BDNF as well as their receptors and caspase-3 in the forebrain. The increased BDNF protein level followed by decreased caspase-3 protein level was probably dependent on body temperature under anoxic conditions and was observed only in rats maintaining decreased body temperature. The positive effect of BDNF was not observed under hyperthermic conditions. Moreover, BDNF level changes correlated with body temperature probably affected the learning and spatial memory in juvenile rats.

Lung injury after asphyxia and hemorrhagic shock in newborn piglets: Analysis of structural and inflammatory changes.

OBJECTIVE: Asphyxia of newborns is a severe and frequent challenge of the peri- and postnatal period. The purpose of this study was to study early morphological, immunological and structural alterations in lung tissue after asphyxia and hemorrhage (AH). METHODS: 44 neonatal piglets (age 32 hrs) underwent asphyxia and hemorrhage (AH) and were treated according to the international liaison committee of resuscitation (ILCOR) guidelines. For this study, 15 piglets (blood transfusion (RBC) n = 9; NaCl n = 6, mean age 31 hrs) were randomly picked. 4 hours after ROSC (return of spontaneous circulation), lung tissue and blood samples were collected. RESULTS: An elevation of myeloperoxidase (MPO) activity was observed 4 hrs after AH accompanied by an increase of surfactant D after RBC treatment. After AH tight junction proteins Claudin 18 and junctional adhesion molecule 1 (JAM1) were down-regulated, whereas Occludin was increased. Furthermore, after AH and RBC treatment dephosphorylated active form of Connexin 43 was increased. CONCLUSIONS: AH in neonatal pigs is associated with early lung injury, inflammation and alterations of tight junctions (Claudin, Occludin, JAM-1) and gap junctions (Connexin 43) in lung tissue, which contributes to the development of lung edema and impaired function.

The Long-Term Impairment in Redox Homeostasis Observed in the Hippocampus of Rats Subjected to Global Perinatal Asphyxia (PA) Implies Changes in Glutathione-Dependent Antioxidant Enzymes and TIGAR-Dependent Shift Towards the Pentose Phosphate Pathways: Effect of Nicotinamide.

We have recently reported that global perinatal asphyxia (PA) induces a regionally sustained increase in oxidized glutathione (GSSG) levels and GSSG/GSH ratio, a decrease in tissue-reducing capacity, a decrease in catalase activity, and an increase in apoptotic caspase-3-dependent cell death in rat neonatal brain up to 14 postnatal days, indicating a long-term impairment in redox homeostasis. In the present study, we evaluated whether the increase in GSSG/GSH ratio observed in hippocampus involves changes in glutathione reductase (GR) and glutathione peroxidase (GPx) activity, the enzymes reducing glutathione disulfide (GSSG) and hydroperoxides, respectively, as well as catalase, the enzyme protecting against peroxidation. The study also evaluated whether there is a shift in the metabolism towards the penthose phosphate pathway (PPP), by measuring TIGAR, the TP53-inducible glycolysis and apoptosis regulator, associated with delayed cell death, further monitoring calpain activity, involved in bax-dependent cell death, and XRCC1, a scaffolding protein interacting with genome sentinel proteins. Global PA was induced by immersing fetus-containing uterine horns removed by a cesarean section from on term rat dams into a water bath at 37 °C for 21 min. Asphyxia-exposed and sibling cesarean-delivered fetuses were manually resuscitated and nurtured by surrogate dams. Animals were euthanized at postnatal (P) days 1 or 14, dissecting samples from hippocampus to be assayed for glutathione, GR, GPx (all by spectrophotometry), catalase (Western blots and ELISA), TIGAR (Western blots), calpain (fluorescence), and XRCC1 (Western blots). One hour after delivery, asphyxia-exposed and control neonates were injected with either 100 µl saline or 0.8 mmol/kg nicotinamide, i.p., shown to protect from the short- and long-term consequences of PA. It was found that global PA produced (i) a sustained increase of GSSG levels and GSSG/GSH ratio at P1 and P14; (ii) a decrease of GR, GPx, and catalase activity at P1 and P14; (iii) a decrease at P1, followed by an increase at P14 of TIGAR levels; (iv) an increase of calpain activity at P14; and (v) an increase of XRCC1 levels, but only at P1. (vi) Nicotinamide prevented the effect of PA on GSSG levels and GSSG/GSH ratio, and on GR, GPx, and catalase activity, also on increased TIGAR levels and calpain activity observed at P14. The present study demonstrates that the long-term impaired redox homeostasis observed in the hippocampus of rats subjected to global PA implies changes in GR, GPx, and catalase, and a shift towards PPP, as indicated by an increase of TIGAR levels at P14.

Brain oxidative damage in murine models of neonatal hypoxia/ischemia and reoxygenation.

The brain is one of the main organs affected by hypoxia and reoxygenation in the neonatal period and one of the most vulnerable to oxidative stress. Hypoxia/ischemia and reoxygenation leads to impairment of neurogenesis, disruption of cortical migration, mitochondrial damage and neuroinflammation. The extent of the injury depends on the clinical manifestation in the affected regions. Preterm newborns are highly vulnerable, and they exhibit severe clinical manifestations such as intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP) and diffuse white matter injury (DWMI) among others. In the neonatal period, the accumulation of high levels of reactive oxygen species exacerbated by the immature antioxidant defense systems in represents cellular threats that, if they exceed or bypass physiological counteracting mechanisms, are responsible of significant neuronal damage. Several experimental models in mice mimic the consequences of perinatal asphyxia and the use of oxygen in the reanimation process that produce brain injury. The aim of this review is to highlight brain damage associated with oxidative stress in different murine models of hypoxia/ischemia and reoxygenation.

Novel interventions to reduce oxidative-stress related brain injury in neonatal asphyxia.

Perinatal asphyxia-induced brain injury may present as hypoxic-ischemic encephalopathy in the neonatal period, and disability including cerebral palsy in the long term. The brain injury is secondary to both the hypoxic-ischemic event and the reoxygenation-reperfusion following resuscitation. Early events in the cascade of brain injury can be classified as either inflammation or oxidative stress through the generation of free radicals. The objective of this paper is to present efforts that have been made to limit the oxidative stress associated with hypoxic-ischemic encephalopathy. In the acute phase of ischemia/hypoxia and reperfusion/reoxygenation, the outcomes of asphyxiated infants can be improved by optimizing the initial delivery room stabilization. Interventions include limiting oxygen exposure, and shortening the time to return of spontaneous circulation through improved methods for supporting hemodynamics and ventilation. Allopurinol, melatonin, noble gases such as xenon and argon, and magnesium administration also target the acute injury phase. Therapeutic hypothermia, N-acetylcysteine2-iminobiotin, remote ischemic postconditioning, cannabinoids and doxycycline target the subacute phase. Erythropoietin, mesenchymal stem cells, topiramate and memantine could potentially limit injury in the repair phase after asphyxia. To limit the injurious biochemical processes during the different stages of brain injury, determination of the stage of injury in any particular infant remains essential. Currently, therapeutic hypothermia is the only established treatment in the subacute phase of asphyxia-induced brain injury. The effects and side effects of oxidative stress reducing/limiting medications may however be difficult to predict in infants during therapeutic hypothermia. Future neuroprotection in asphyxiated infants may indeed include a combination of therapies. Challenges include timing, dosing and administration route for each neuroprotectant.

Neuroprotective strategies following perinatal hypoxia-ischemia: Taking aim at NOS.

Perinatal asphyxia is characterized by oxygen deprivation and lack of perfusion in the perinatal period, leading to hypoxic-ischemic encephalopathy and sequelae such as cerebral palsy, mental retardation, cerebral visual impairment, epilepsy and learning disabilities. On cellular level PA is associated with a decrease in oxygen and glucose leading to ATP depletion and a compromised mitochondrial function. Upon reoxygenation and reperfusion, the renewed availability of oxygen gives rise to not only restoration of cell function, but also to the activation of multiple detrimental biochemical pathways, leading to secondary energy failure and ultimately, cell death. The formation of reactive oxygen species, nitric oxide and peroxynitrite plays a central role in the development of subsequent neurological damage. In this review we give insight into the pathophysiology of perinatal asphyxia, discuss its clinical relevance and summarize current neuroprotective strategies related to therapeutic hypothermia, ischemic postconditioning and pharmacological interventions. The review will also focus on the possible neuroprotective actions and molecular mechanisms of the selective neuronal and inducible nitric oxide synthase inhibitor 2-iminobiotin that may represent a novel therapeutic agent for the treatment of hypoxic-ischemic encephalopathy, both in combination with therapeutic hypothermia in middle- and high-income countries, as well as stand-alone treatment in low-income countries.