Publications by authors named "Rønnaug Solberg"

32 Publications

Quantification of circulating cell-free DNA (cfDNA) in urine using a newborn piglet model of asphyxia.

PLoS One 2019 31;14(12):e0227066. Epub 2019 Dec 31.

Department of Pediatric Research, Division of Pediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway.

Cell free DNA (cfDNA) in plasma has been described as a potential diagnostic indicator for a variety of clinical conditions, including neonatal hypoxia. Neonatal hypoxia or perinatal asphyxia is a severe medical condition caused by a temporary interruption in oxygen availability during birth. Previously, we have reported temporal changes of cfDNA detected in blood in a newborn piglet model of perinatal asphyxia. However, cfDNA can also be found in other body liquids, opening for a less invasive diagnostic prospective. The objective of this study was to test and establish a reliable method for the isolation and quantification of cfDNA from urine and to explore changes in the quantities of cfDNA using a newborn piglet model of asphyxia. Animals were exposed to hypoxia-reoxygenation (n = 6), hypoxia-reoxygenation + hypothermia (n = 6) or were part of the sham-operated control group (n = 6) and urine samples (n = 18) were collected at 570 minutes post-intervention. Two alternative applications of cfDNA measurement were tested, an indirect method comprising a centrifugation step together with DNA extraction with magnetic beads versus a direct assessment based on two centrifugation steps. CfDNA concentrations were determined by a fluorescent assay using PicoGreen and by qRT-PCR. Genomic (gDNA) and mitochondrial DNA (mtDNA) cfDNA were determined in parallel, taking into account potential differences in the rates of damages caused by oxidative stress. In contrast to previous publications, our results indicate that the direct method is insufficient. Application of the indirect method obtained with the fluorescence assay revealed mean cfDNA levels (SD) of 1.23 (1.76) ng/ml for the hypoxia samples, 4.47 (6.15) ng/ml for the samples exposed to hypoxia + hypothermia and 2.75 (3.62) ng/ml for the control animals. The mean cfDNA levels in piglets exposed to hypoxia + hypothermia revealed significantly higher cfDNA amounts compared to mean cfDNA levels in the samples purely exposed to hypoxia (p < 0.05); however, no significant difference could be determined when compared to the control group (p = 0.09). Application of the indirect method by qRT-PCR revealed mean cfDNA levels of mtDNA and gDNA at the detection limit of the technique and thus no reliable statistics could be performed between the observed cfDNA levels in the investigated groups. The methodology for detection and monitoring of cfDNA in urine has to be further optimized before it can be applied in a clinical setting in the future.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227066PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938324PMC
April 2020

Temporal patterns of circulating cell-free DNA (cfDNA) in a newborn piglet model of perinatal asphyxia.

PLoS One 2018 26;13(11):e0206601. Epub 2018 Nov 26.

Department of Pediatric Research, Division of Pediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway.

Perinatal asphyxia is a severe medical condition resulting from oxygen deficiency (hypoxia) at the time of birth, causing worldwide approximately 680,000 newborn deaths every year. Better prediction of severity of damages including early biomarkers is highly demanded. Elevated levels of circulating cell-free DNA (cfDNA) in blood have been reported for a range of different diseases and conditions, including cancer and prematurity. The objective of this study was to validate methods for assessing cfDNA in blood and cerebrospinal fluid (CSF) and to explore temporal variations in a piglet model of neonatal hypoxia-reoxygenation. Different cfDNA extraction methods in combination with cfDNA detection systems were tested, including a fluorescent assay using SYBR Gold and a qRT-PCR-based technique. Newborn piglets (n = 55) were exposed to hypoxia-reoxygenation, hypoxia-reoxygenation and hypothermia, or were part of the sham-operated control group. Blood was sampled at baseline and at post-intervention, further at 30, 270, and 570 minutes after the end of hypoxia. Applying the fluorescent method, cfDNA concentration in piglets exposed to hypoxia (n = 32) increased from 36.8±27.6 ng/ml prior to hypoxia to a peak level of 61.5±54.9 ng/ml after the intervention and deceased to 32.3±19.1 ng/ml at 570 minutes of reoxygenation, whereas the group of sham-operated control animals (n = 11) revealed a balanced cfDNA profile. Animals exposed to hypoxia and additionally treated with hypothermia (n = 12) expressed a cfDNA concentration of 54.4±16.9 ng/ml at baseline, 39.2±26.9 ng/ml at the end of hypoxia, and of 41.1±34.2 ng/ml at 570 minutes post-intervention. Concentrations of cfDNA in the CSF of piglets exposed to hypoxia revealed at post-intervention higher levels in comparison to the controls. However, these observations were only tendencies and not significant. In a first methodological proof-of-principle study exploring cfDNA using a piglet model of hypoxia-reoxygenation variations in the temporal patterns suggest that cfDNA might be an early indicator for damages caused by perinatal asphyxia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206601PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261042PMC
April 2019

Dynamic TSPO-PET for assessing early effects of cerebral hypoxia and resuscitation in new born pigs.

Nucl Med Biol 2018 11 24;66:49-57. Epub 2018 Aug 24.

Div. of Radiology and Nuclear Medicine, Oslo University Hospital, PO box 4950, Nydalen, N-0424 Oslo, Norway; Faculty of Medicine, University of Oslo, PO Box1078, Blindern, N-0316 Oslo, Norway.

Introduction: Inflammation associated with microglial activation may be an early prognostic indicator of perinatal hypoxic ischemic injury, where translocator protein (TSPO) is a known inflammatory biomarker. This piglet study used dynamic TSPO-PET with [F]GE180 to evaluate if microglial activation after global perinatal hypoxic injury could be detected.

Methods: New born anesthetized pigs (n = 14) underwent hypoxia with fraction of inspired oxygen (FiO)0.08 until base excess -20 mmol/L and/or a mean arterial blood pressure decrease to 20 mm Hg, followed by resuscitation with FiO 0.21 or 1.0. Three piglets served as controls and one had intracranial injection of lipopolysaccharide (LPS). Whole body [F]GE180 Positron emission tomography-computed tomography (PET-CT) was performed repeatedly up to 32 h after hypoxia and resuscitation. Volumes of interest were traced in the basal ganglia, cerebellum and liver using MRI as anatomic correlation. Standardized uptake values (SUVs) were measured at baseline and four time-points, quantifying microglial activity over time. Statistical analysis used Mann Whitney- and Wilcoxon rank test with significance value set to p < 0.05.

Results: At baseline (n = 5), mean SUVs ±1 standard deviation were 0.43 ± 0.10 and 1.71 ± 0.62 in brain and liver respectively without significant increase after hypoxia at the four time-points (n = 5-13/time point). Succeeding LPS injection, SUV increased 80% from baseline values.

Conclusions: Cerebral inflammatory response caused by severe asphyxia was not possible to detect with [F]GE180 PET CT the first 32 h after hypoxia and only sparse hepatic uptake was revealed.

Advances In Knowledge: Early microglial activation as indicator of perinatal hypoxic ischemic injury was not detectable by TSPO-PET with [F]GE180.

Implications For Patient Care: TSPO-PET with [F]GE180 might not be suitable for early detection of perinatal hypoxic ischemic brain injury.
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http://dx.doi.org/10.1016/j.nucmedbio.2018.08.004DOI Listing
November 2018

Regional differences of hypothermia on oxidative stress following hypoxia-ischemia: a study of DHA and hypothermia on brain lipid peroxidation in newborn piglets.

J Perinat Med 2018 Dec;47(1):82-89

Department of Pediatric Research, Institute of Surgical Research, University of Oslo, Oslo University Hospital Rikshospitalet, Oslo, Norway.

Background Oxidative stress plays an important part in the pathophysiology of hypoxic-ischemic encephalopathy (HIE) and is reliably measured through prostanoids following lipid peroxidation of polyunsaturated fatty acids (PUFAs). The aim of the study is to measure oxidative stress in the prefrontal cortex, white matter and hippocampus in the brains of hypoxic-ischemic piglets treated with docosahexaenoic acid (DHA) and therapeutic hypothermia (TH) and investigate the additive effects of DHA on hypothermia by factorial design. Methods Fifty-five piglets were randomized as having severe global hypoxia (n=48) or not (sham, n=7). Hypoxic piglets were further randomized: vehicle (VEH), DHA, VEH+hypothermia (HT) or HT+DHA. A total of 5 mg/kg DHA was given intravenously 210 min after the end of hypoxia. Brain tissues were analyzed using liquid chromatography triple quadrupole mass spectrometry technique (LC-MS). A two-way analysis of variance (ANOVA) was performed with DHA and HT as main effects. Results In the white matter, we found main effects of DHA on DH-isoprostanes (P=0.030) and a main effect of HT on F4-neuroprostanes (F4-NeuroPs) (P=0.007), F2-isoprostanes (F2-IsoPs) (P=0.043) and DH-isoprostanes (P=0.023). In the cortex, the ANOVA analysis showed the interactions of main effects between DHA and HT for neurofuranes (NeuroFs) (P=0.092) and DH-isoprostanes (P=0.015) as DHA significantly reduced lipid peroxidation in the absence of HT. DHA compared to VEH significantly reduced NeuroFs (P=0.019) and DH-isoprostanes (P=0.010). No differences were found in the hippocampus. Conclusion After severe hypoxia, HT reduced lipid peroxidation in the white matter but not in the cortical gray matter. HT attenuated the reducing effect of DHA on lipid peroxidation in the cortex. Further studies are needed to determine whether DHA can be an effective add-on therapy for TH.
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http://dx.doi.org/10.1515/jpm-2017-0355DOI Listing
December 2018

DHA and therapeutic hypothermia in a short-term follow-up piglet model of hypoxia-ischemia: Effects on H+MRS biomarkers.

PLoS One 2018 7;13(8):e0201895. Epub 2018 Aug 7.

Department of Pediatric Research, Women and Children's Division and Institute for Surgical Research, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

Background: Therapeutic hypothermia has become the standard of care for newborns with hypoxic-ischemic encephalopathy in high and middle income countries. Docosahexaenoic acid (DHA) has neuroprotective properties of reducing excitotoxicity, neuroinflammation and apoptosis in rodent models. We aim to study whether post hypoxic administration of i.v. DHA will reduce H+MRS biomarkers and gene expression of inflammation and apoptosis both with and without hypothermia in a large animal model.

Methods: Fifty-five piglets were randomized to severe global hypoxia (N = 48) or not (Sham, N = 7). Hypoxic piglets were further randomized by factorial design: Vehicle (VEH), DHA, VEH + Hypothermia (HT), or DHA + HT. 5 mg/kg DHA was given intravenously 210 min after end of hypoxia. Two-way ANOVA analyses were performed with DHA and hypothermia as main effects.

Results: Cortical lactate/N-acetylaspartate (Lac/NAA) was significantly reduced in DHA + HT compared to HT. DHA had significant main effects on increasing N-acetylaspartate and glutathione in hippocampus. Therapeutic hypothermia significantly reduced the Lac/NAA ratio and protein expression of IL-1β and TNFα in hippocampus and reduced Troponin T in serum. Neuropathology showed significant differences between sham and hypoxia, but no differences between intervention groups.

Conclusion: DHA and therapeutic hypothermia significantly improve specific H+MRS biomarkers in this short-term follow up model of hypoxia-ischemia. Longer recovery periods are needed to evaluate whether DHA can offer translational neuroprotection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201895PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080779PMC
February 2019

Combined Inhibition of C5 and CD14 Attenuates Systemic Inflammation in a Piglet Model of Meconium Aspiration Syndrome.

Neonatology 2018 27;113(4):322-330. Epub 2018 Feb 27.

Department of Immunology, Oslo University Hospital and K.G. Jebsen IRC, University of Oslo, Oslo, Norway.

Background: Meconium aspiration syndrome (MAS) is a severe lung condition affecting newborns and it can lead to a systemic inflammatory response. We previously documented complement activation and cytokine release in a piglet MAS model. Additionally, we showed ex vivo that meconium-induced inflammation was dependent on complement and Toll-like receptors.

Objectives: To assess the efficacy of the combined inhibition of complement (C5) and CD14 on systemic inflammation induced in a forceful piglet MAS model.

Methods: Thirty piglets were randomly allocated to a treatment group receiving the C5-inhibitor SOBI002 and anti-CD14 (n = 15) and a nontreated control group (n = 15). MAS was induced by intratracheal meconium instillation, and the piglets were observed for 5 h. Complement, cytokines, and myeloperoxidase (MPO) were measured by ELISA.

Results: SOBI002 ablated C5 activity and the formation of the terminal complement complex in vivo. The combined inhibition attenuated the inflammasome cytokines IL-1β and IL-6 by 60 (p = 0.029) and 44% (p = 0.01), respectively, and also MPO activity in the bronchoalveolar fluid by 42% (p = 0.017). Ex vivo experiments in human blood revealed that the combined regimen attenuated meconium-induced MPO release by 64% (p = 0.008), but there was only a negligible effect with single inhibition, indicating a synergic cross-talk between the key molecules C5 and CD14.

Conclusion: Combined inhibition of C5 and CD14 attenuates meconium-induced inflammation in vivo and this could become a future therapeutic regimen for MAS.
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http://dx.doi.org/10.1159/000486542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008878PMC
September 2019

DHA reduces oxidative stress following hypoxia-ischemia in newborn piglets: a study of lipid peroxidation products in urine and plasma.

J Perinat Med 2018 Feb;46(2):209-217

Department of Pediatric Research, Institute of Surgical Research, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

Background: Lipid peroxidation mediated by reactive oxygen species is a major contributor to oxidative stress. Docosahexaenoic acid (DHA) has anti-oxidant and neuroprotective properties. Our objective was to assess how oxidative stress measured by lipid peroxidation was modified by DHA in a newborn piglet model of hypoxia-ischemia (HI).

Methods: Fifty-five piglets were randomized to (i) hypoxia, (ii) DHA, (iii) hypothermia, (iv) hypothermia+DHA or (v) sham. All groups but sham were subjected to hypoxia by breathing 8% O2. DHA was administered 210 min after end of hypoxia and the piglets were euthanized 9.5 h after end of hypoxia. Urine and blood were harvested at these two time points and analyzed for F4-neuroprostanes, F2-isoprostanes, neurofuranes and isofuranes using UPLC-MS/MS.

Results: F4-neuroprostanes in urine were significantly reduced (P=0.006) in groups receiving DHA. Hypoxia (median, IQR 1652 nM, 610-4557) vs. DHA (440 nM, 367-738, P=0.016) and hypothermia (median, IQR 1338 nM, 744-3085) vs. hypothermia+DHA (356 nM, 264-1180, P=0.006). The isoprostane compound 8-iso-PGF2α was significantly lower (P=0.011) in the DHA group compared to the hypoxia group. No significant differences were found between the groups in blood.

Conclusion: DHA significantly reduces oxidative stress by measures of lipid peroxidation following HI in both normothermic and hypothermic piglets.
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http://dx.doi.org/10.1515/jpm-2016-0334DOI Listing
February 2018

High-Dose Cannabidiol Induced Hypotension after Global Hypoxia-Ischemia in Piglets.

Neonatology 2017 1;112(2):143-149. Epub 2017 Jun 1.

Division of Pediatric and Adolescent Medicine, Department of Pediatric Research, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

Background: Cannabidiol (CBD) is considered a promising neuroprotectant after perinatal hypoxia-ischemia (HI). We have previously studied the effects of CBD 1 mg/kg in the early phase after global HI in piglets. In contrast to prior studies, we found no evidence of neuroprotection and hypothesized that higher doses might be required to demonstrate efficacy in this animal model.

Objective: To assess the safety and potential neuroprotective effects of high-dose CBD.

Methods: Anesthetized newborn piglets underwent global HI by ventilation with 8% O2 until the point of severe metabolic acidosis (base excess -20 mmol/L) and/or hypotension (mean arterial blood pressure ≤20 mm Hg). Piglets were randomized to intravenous treatment with vehicle (n = 9) or CBD (n = 13). The starting dose, CBD 50 mg/kg, was reduced if adverse effects occurred. The piglets were euthanized 9.5 h after HI and tissue was collected for analysis.

Results: CBD 50 mg/kg (n = 4) induced significant hypotension in 2 out of 4 piglets, and 1 out of 4 piglets suffered a fatal cardiac arrest. CBD 25 mg/kg (n = 4) induced significant hypotension in 1 out of 4 piglets, while 10 mg/kg (n = 5) was well tolerated. A significant negative correlation between the plasma concentration of CBD and hypotension during drug infusion was observed (p < 0.005). Neuroprotective effects were evaluated in piglets that did not display significant hypotension (n = 9) and CBD did not alter the degree of neuronal damage as measured by a neuropathology score, levels of the astrocytic marker S100B in CSF, magnetic resonance spectroscopy markers (Lac/NAA and Glu/NAA ratios), or plasma troponin T.

Conclusions: High-dose CBD can induce severe hypotension and did not offer neuroprotection in the early phase after global HI in piglets.
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http://dx.doi.org/10.1159/000471786DOI Listing
May 2018

Plasma metabolite score correlates with Hypoxia time in a newly born piglet model for asphyxia.

Redox Biol 2017 08 7;12:1-7. Epub 2017 Feb 7.

Neonatal Research Group, Health Research Institute Hospital La Fe, Avenida Fernando Abril Martorell 106, Valencia, Spain; Division of Neonatology, University & Polytechnic Hospital La Fe, Avenida Fernando Abril Martorell 106, Valencia, Spain. Electronic address:

Hypoxic-ischemic encephalopathy (HIE) secondary to perinatal asphyxia is a leading cause of mortality and acquired long-term neurologic co-morbidities in the neonate. The most successful intervention for the treatment of moderate to severe HIE is moderate whole body hypothermia initiated within 6h from birth. The objective and prompt identification of infants who are at risk of developing moderate to severe HIE in the critical first hours still remains a challenge. This work proposes a metabolite score calculated based on the relative intensities of three metabolites (choline, 6,8-dihydroxypurine and hypoxanthine) that showed maximum correlation with hypoxia time in a consolidated piglet model for neonatal hypoxia-ischemia. The metabolite score's performance as a biomarker for perinatal hypoxia and its usefulness for clinical grading and decision making have been assessed and compared to the performance of lactate which is currently considered the gold standard. For plasma samples withdrawn before and directly after a hypoxic insult, the metabolite score performed similar to lactate. However, it provided an enhanced predictive capacity at 2h after resuscitation. The present study evidences the usefulness of the metabolite score for improving the early assessment of the severity of the hypoxic insult based on serial determinations in a minimally invasive biofluid. The applicability of the metabolite score for clinical diagnosis and patient stratification for hypothermia treatment has to be confirmed in multicenter trials involving newborns suffering from HIE.
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http://dx.doi.org/10.1016/j.redox.2017.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310173PMC
August 2017

DHA Reduces Oxidative Stress after Perinatal Asphyxia: A Study in Newborn Piglets.

Neonatology 2017 1;112(1):1-8. Epub 2017 Feb 1.

Department of Pediatric Research, University of Oslo, Oslo University Hospital Rikshospitalet, Oslo, Norway.

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.
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http://dx.doi.org/10.1159/000454982DOI Listing
April 2018

Assessment of phospholipid synthesis related biomarkers for perinatal asphyxia: a piglet study.

Sci Rep 2017 01 10;7:40315. Epub 2017 Jan 10.

Neonatal Research Group, Health Research Institute Hospital La Fe, Avenida Fernando Abril Martorell 106, 46026 Valencia, Spain.

The prompt and reliable identification of infants at risk of hypoxic-ischemic encephalopathy secondary to perinatal asphyxia in the first critical hours is important for clinical decision-making and yet still remains a challenge. This work strives for the evaluation of a panel of metabolic biomarkers that have been associated with the hypoxic-ischemic insult in the perinatal period. Plasma and urine samples from a consolidated newborn piglet model of hypoxia and withdrawn before and at different time points after a hypoxic insult were analyzed and compared to a control group. Time-dependent metabolic biomarker profiles were studied and observed patterns were similar to those of lactate levels, which are currently considered the gold standard for assessing hypoxia. Class prediction performance could be improved by the use of a combination of the whole panel of determined metabolites in plasma as compared to lactate values. Using a multivariate model including lactate together with the studied metabolic biomarkers allowed to improve the prediction performance of duration of hypoxia time, which correlates with the degree of brain damage. The present study evidences the usefulness of choline and related metabolites for improving the early assessment of the severity of the hypoxic insult.
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http://dx.doi.org/10.1038/srep40315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223196PMC
January 2017

Short-term effects of cannabidiol after global hypoxia-ischemia in newborn piglets.

Pediatr Res 2016 11 21;80(5):710-718. Epub 2016 Jul 21.

Department of Pediatric Research, Women and Children's Division and Institute for Surgical research, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

Background: Cannabidiol (CBD), a nonpsychoactive cannabinoid, has shown neuroprotective actions after neonatal hypoxia-ischemia (HI) in animals. We wanted to further explore the effects of CBD, alone and in conjunction with hypothermia, in a piglet model of global HI.

Methods: Fifty-five anesthetized newborn piglets were randomized to either controls (n = 7) or HI (n = 48) by ventilation with 8% O until mean arterial blood pressure reached 20 mmHg and/or base excess reached -20 mmol/l. After resuscitation piglets were randomized to either: vehicle (VEH), CBD 1mg/kg, VEH+hypothermia (H) or CBD 1mg/kg+H (each n = 12). Piglets were euthanized 9.5 h after HI and plasma, urine, cerebrospinal fluid, and brain tissue were sampled for analysis.

Results: HI induced global damage with significantly increased neuropathology score, S100B in cerebrospinal fluid, hippocampal proton magnetic resonance spectroscopy biomarkers, plasma troponin-T, and urinary neutrophil gelatinase-associated lipocalin. CBD alone did not have any significant effects on these parameters while CBD+H reduced urinary neutrophil gelatinase-associated lipocalin compared with VEH+H (P < 0.05). Both hypothermic groups had significantly lower glutamate/N-acetylaspartate ratios (P < 0.01) and plasma troponin-T (P<0.05) levels compared with normothermic groups.

Conclusion: In contrast to previous studies, we do not find significant protective effects of CBD after HI in piglets. Evaluation of CBD in higher doses might be warranted.
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http://dx.doi.org/10.1038/pr.2016.149DOI Listing
November 2016

Temporal Profile of Circulating microRNAs after Global Hypoxia-Ischemia in Newborn Piglets.

Neonatology 2017 18;111(2):133-139. Epub 2016 Oct 18.

Division of Pediatric and Adolescent Medicine, Department of Pediatric Research, Oslo University Hospital, Rikshospitalet, Norway.

Background: There is a lack of reliable biomarkers that can identify and grade acute hypoxic-ischemic encephalopathy in newborns. MicroRNAs (miRNA) are short, non-coding strands of RNA that are released into the circulation in response to tissue stress and injury. Some miRNAs are highly tissue specific and thus may potentially be non-invasive biomarkers of neonatal hypoxic-ischemic brain injury.

Objective: The aim of this study was to characterize the temporal expression of selected circulating miRNAs in a clinically relevant piglet model of neonatal hypoxia-ischemia (HI).

Methods: A total of 13 anesthetized newborn piglets were randomized to either a control group (n = 5) or transient global HI group (n = 8). HI was achieved by ventilation with 8% oxygen until the point of severe acidosis (arterial base excess ≤-20 mmol/l) and/or hypotension (mean arterial blood pressure ≤20 mm Hg) was reached. Plasma was sampled at baseline, at the end of HI and 0.5, 3.5 and 9.5 h after HI. MiRNA expression was measured by qRT-PCR.

Results: Compared to baseline, miR-374a increased during HI (p = 0.01), remained elevated at 0.5 h after HI (p = 0.02) and was downregulated at 9.5 h after HI (p = 0.02). MiR-210 increased during HI (p = 0.02) and rapidly normalized by 0.5 h after HI. MiR-124 and miR-125b did not exhibit significant alterations. Correlations were observed between miR-374a, arterial pH, base excess and lactate levels, and between miR-210 and pO2 (p < 0.05).

Conclusions: Our data suggest that miR-374a and miR-210 are important regulators in neonatal HI and might have a place as biomarkers in this setting.
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http://dx.doi.org/10.1159/000449032DOI Listing
December 2017

N-Acetylcysteine Amide Exerts Possible Neuroprotective Effects in Newborn Pigs after Perinatal Asphyxia.

Neonatology 2017 6;111(1):12-21. Epub 2016 Aug 6.

Department of Pediatric Research, University of Oslo, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

Background: Perinatal asphyxia and ensuing reoxygenation change the antioxidant capacity of cells and organs.

Objectives: To analyze the neuroprotective effect of the antioxidant N-acetylcysteine amide (NACA) after perinatal hypoxia-reoxygenation with an emphasis on proinflammatory cytokines and the transcription factor NF-κB in the prefrontal cortex of neonatal pigs.

Methods: Twenty-nine newborn pigs, aged 12-36 h, were subjected to global hypoxia and hypercapnia. One sham-operated group (n = 5) and 2 experimental groups (n = 12) were exposed to 8% oxygen, until the base excess was -20 mmol/l or the mean arterial blood pressure fell to <20 mm Hg (asphyxia with NACA or saline). The pigs were observed for 9.5 h after hypoxia. Samples of prefrontal cortex and plasma were analyzed.

Results: Cortex: there was no significant difference in mRNA expression between the intervention groups regarding IL-1β, IL6, TNFα, MMP2, MMP9 or IL18. Pigs exposed to hypoxia-reoxygenation and treatment with NACA (NACA-pigs) had a significantly lower protein concentration of IL-1β than pigs treated with saline (placebo controls), i.e. 8.8 ± 3.9 versus 16.8 ± 10.5 pg/mg protein (p = 0.02). The activation of the transcription factor NF-κB (measured as the fold-change of phosphorylated p65Ser 536), was reduced in the NACA-pigs when compared to the placebo controls (5.2 ± 4.3 vs. 16.0 ± 13.5; p = 0.02). No difference between the intervention groups regarding brain histopathology or in the levels of 8-oxoguanine measured in the prefrontal cortex were observed. Plasma: the NACA-pigs had a stronger reduction of TNFα in the first 30 min following asphyxia compared with the placebo controls, i.e. 36 (30-44) versus 24 (14-32)% (p = 0.01).

Conclusion: The reduced levels of the pivotal inflammatory markers IL-1β and TNFα and the transcription factor NF-κB may indicate that NACA has possible neuroprotective effects after perinatal asphyxia.
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http://dx.doi.org/10.1159/000447255DOI Listing
December 2017

Changes of the plasma metabolome of newly born piglets subjected to postnatal hypoxia and resuscitation with air.

Pediatr Res 2016 08 7;80(2):284-92. Epub 2016 Apr 7.

Neonatal Research Group, Health Research Institute Hospital La Fe, Valencia, Spain.

Background: Perinatal hypoxic-ischemic brain damage is a major cause of mortality and morbidity in the neonatal period. Currently, limited ranges of biochemical tests assessing the intensity and duration of hypoxia are ready for clinical use. However, the need to initiate hypothermia therapy early after the clinical suspicion of hypoxic-ischemic encephalopathy requires the availability of early and reliable hypoxia markers. We have sought these biomarkers in an experimental model of hypoxia reoxygenation.

Methods: Hypoxia and hypotension were induced in newborn piglets following a standardized model and reoxygenation was carried out using room air (RA). An untargeted liquid chromatography-time of flight mass spectrometry (LC-TOFMS) approach was used to assess changes in the metabolomic profile of plasma samples after intense hypoxia and upon reoxygenation.

Results: At the end of hypoxia, the plasma metabolome showed an increased plasma concentration of analytes reflecting a metabolic adaptation to prolonged anaerobiosis. However, after resuscitation, metabolite levels returned to the starting values.

Conclusion: Severe hypoxia induces early, significant, and transient changes of specific metabolites in the plasma metabolome, which represent a snapshot of the biochemical adaptation of mammals to intense hypoxia. These metabolites could have applicability in predicting the severity of hypoxia in the clinical setting.
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http://dx.doi.org/10.1038/pr.2016.66DOI Listing
August 2016

Protein-bound tyrosine oxidation, nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry.

Anal Chim Acta 2016 Mar 3;913:104-10. Epub 2016 Feb 3.

Neonatal Research Group, Health Research Institute La Fe, Avenida Fernando Abril Martorell 101, 46026, Valencia, Spain. Electronic address:

Background: Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions.

Objective: To develop, validate and apply a rapid method for the quantification of known hallmarks of tyrosine oxidation, nitration and chlorination in plasma and tissue proteins providing a snapshot of the oxidative stress and inflammatory status of the organism and of target organs respectively.

Material And Methods: The extraction and clean up procedure entailed protein precipitation, followed by protein re-suspension and enzymatic digestion with pronase. An Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method was developed to quantify protein released ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3NO2-Tyr) and 3-chlorotyrosine (3Cl-Tyr) as well as native phenylalanine (Phe) and tyrosine (p-Tyr) in plasma and tissue from a validated hypoxic newborn piglet experimental model.

Results: In plasma there was a significant increase in the 3NO2-Tyr/p-Tyr ratio. On the other hand m-Tyr/Phe and 3Cl-Tyr/p-Tyr ratios were significantly increased in liver of hypoxic compared with normoxic animals. Although no significant differences were found in brain tissue, a clear tendency to increased ratios was observed under hypoxic conditions.

Conclusions: UPLC-MS/MS has proven suitable for the analysis of plasma and tissue samples from newborn piglets. The analysis of biomarkers of protein oxidation, nitration and chlorination will be applied in future studies aiming to provide a deeper insight into the mechanisms of oxidation-derived protein modification caused during neonatal asphyxia and resuscitation.
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http://dx.doi.org/10.1016/j.aca.2016.01.054DOI Listing
March 2016

Development of a reliable method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to measure thiol-associated oxidative stress in whole blood samples.

J Pharm Biomed Anal 2016 May 9;123:104-12. Epub 2016 Feb 9.

Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain. Electronic address:

The aminothiols are biological compounds with numerous vital functions. One of the most relevant is their role as antioxidant systems. The reduced to oxidized ratios are extremely useful indicators of oxidative stress and cellular redox status. We have validated an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to determine the levels of glutathione, cysteine, homocysteine, and their respective oxidized compounds in whole blood samples. Results showed excellent linearity for all the analytes with correlation coefficients between 0.990 and 0.997, suitable precision with intra-day coefficient of variation ≤20%, and satisfactory accuracy with recoveries between 75 and 130%. The limits of detection in whole blood samples were 1.16 nmol L(-1) for glutathione, 115.8 nmol L(-1) for oxidized glutathione, 9.3 nmol L(-1) for homocystine, 92.6 nmol L(-1) for homocysteine, 347 nmol L(-1) for cystine and 0.23 nmol L(-1) for cysteine. The suitability of the method was ascertained in whole blood samples (n=80) from a consolidated experimental model of hypoxia-reoxygenation in newborn piglets.
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http://dx.doi.org/10.1016/j.jpba.2016.02.007DOI Listing
May 2016

Perinatal Asphyxia May Influence the Level of Beta-Amyloid (1-42) in Cerebrospinal Fluid: An Experimental Study on Newborn Pigs.

PLoS One 2015 26;10(10):e0140966. Epub 2015 Oct 26.

Dept of Pediatric Research, Oslo University Hospital, Oslo, Norway.

Objective: Total tau (T-tau), phosphorylated tau (p-Tau) and Beta-Amyloid 1-42 (AB42) in Cerebrospinal Fluid (CSF) are useful biomarkers in neurodegenerative diseases. The aim of the study was to investigate the role of these and other CSF biomarkers (T-tau, p-Tau, AB42, S100B and NSE), during hypoxia-reoxygenation in a newborn pig model.

Design: Thirty newborn pigs were included in a study of moderate or severe hypoxia. The moderate hypoxia group (n = 12) was exposed to global hypoxia (8% O2) until Base excess (BE) reached -15 mmol/l. The pigs in the group exposed to severe hypoxia (n = 12) received 8% O2 until BE reached -20 mmol/l or mean Blood Pressure fell below 20 mm Hg, The control group (n = 6) was kept at room air. For all treatments, the CSF was collected at 9.5 hours after the intervention.

Results: The level of AB42 in CSF was significantly lower in the pigs exposed to severe hypoxia compared with the control group, 922(SD +/-445)pg/ml versus. 1290(SD +/-143) pg/ml (p<0.05), respectively. Further, a non-significant reduction of AB42 was observed in the group exposed to moderate hypoxia T-tau and p-Tau revealed no significant differences between the intervention groups and the control group, however a significantly higher level of S100B was seen in the CSF of pigs receiving hypoxia in comparison to the level in the control group. Further on, there was a moderate negative correlation between the levels of AB42 and S100B in CSF, as well as a moderate negative correlation between Lactate in blood at end of hypoxia and AB42 in CSF.

Interpretation: This is the first study to our knowledge that demonstrated a significant drop in AB42 in CSF after neonatal hypoxia. Whether or not this has an etiological basis for adult neurodegenerative disorders needs to be studied with additional experiments and epidemiological studies. AB42 and S100B are significantly changed in neonatal pigs subjected to hypoxia compared to controls and thus may be valuable biomarkers of perinatal asphyxia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140966PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621034PMC
June 2016

Metabolic adaptation and neuroprotection differ in the retina and choroid in a piglet model of acute postnatal hypoxia.

Pediatr Res 2014 Aug 12;76(2):127-34. Epub 2014 May 12.

Department of Pediatric Research, Institute for Surgical Research, Oslo University Hospital-Rikshospitalet, Oslo, Norway.

Background: Hypoxic-ischemic insults to the neonatal brain may cause neurodevelopmental disorders. Vulnerability of different areas of the neural tissue to hypoxic-ischemic stress might be explained by either heterogeneous sensitivity to oxygen or neuroprotective capability. Our understanding of regional heterogeneity is still incomplete in terms of metabolic reconfiguration and/or activation of neuroprotective mechanisms.

Methods: We studied, by western blotting, reverse-transcriptase PCR, and tandem mass spectrometry, the response of retina and choroid at protein, gene, and metabolic levels during hypoxia in a piglet model of acute postnatal hypoxia.

Results: We evidenced a metabolic shift towards glycolysis in choroid after hypoxia while retina experienced a dramatic energy stress with decreased mitochondrial metabolites. Hypoxia-inducible transcription factor-1α (HIF-1α) was not stabilized in retina during hypoxia, supported by a deficient signaling from v-akt murine thymoma viral oncogene (AKT) and ERK1/2, and unchanged glutathione redox status. In retina, but not in choroid, phosphorylation of p65 (NF-κB) and increased transcription of target genes may have a major role during hypoxic stress.

Conclusion: We showed that the retina engages a distinct pattern of signaling and transcriptional events than observed in the choroid. Retina and choroid may reflect regional sensitivity to hypoxia. While prolonged and intense hypoxia may jeopardize retinal cell survival, choroid sets up a different pattern of response, which promotes adaptation to these adverse conditions.
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http://dx.doi.org/10.1038/pr.2014.70DOI Listing
August 2014

Metabolomic Analysis of the Effect of Postnatal Hypoxia on the Retina in a Newly Born Piglet Model.

PLoS One 2013 18;8(6):e66540. Epub 2013 Jun 18.

Department of Pediatric Research, Institute for Surgical Research, Oslo University Hospital - Rikshospitalet, Oslo, Norway.

The availability of reliable biomarkers of brain injury secondary to birth asphyxia could substantially improve clinical grading, therapeutic intervention strategies, and prognosis. In this study, changes in the metabolome of retinal tissue caused by profound hypoxia in an established neonatal piglet model were investigated using an ultra performance liquid chromatography - quadrupole time of flight mass spectrometry (UPLC-QTOFMS) untargeted metabolomic approach, which included Partial Least Squares - Discriminant Analysis (PLSDA) multivariate data analysis. The initial identification of a set of discriminant metabolites from UPLC-QTOFMS data was confirmed by target UPLC-MS/MS and allowed the selection of endogenous CDP-choline as a promising candidate biomarker for hypoxia-derived brain damage assessing intensity of retinal hypoxia. Results from this study will foster further research on CDP-choline changes occurring during resuscitation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066540PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688918PMC
October 2017

Oxygenation of the newborn: a molecular approach.

Neonatology 2012 1;101(4):315-25. Epub 2012 Jun 1.

Department of Pediatric Research, Women and Children's Division, Oslo University Hospital, University of Oslo, Oslo, Norway.

In this review oxygenation and hyperoxic injury of newborn infants are described through molecular and genetic levels. Protection and repair mechanisms that may be important for a new understanding of oxidative stress in the newborn are discussed. The research summarized in this article represents a basis for the reduced oxygen supplementation and oxidative load of newborn babies, especially since the turn of the century. The mechanisms discussed may also contribute to an understanding of why hyperoxic resuscitation of the newborn may damage DNA and affect its repair, thus increasing the risk that it may be carcinogenic. Today, term babies should be resuscitated with air rather than 100% oxygen and very and extremely low birth weight infants in need of stabilization or resuscitation at birth should be administered initially 21-30% oxygen and the level should be titrated according to the response, preferably measured by pulse oximetry. In the postnatal period the oxygen saturation should be targeted low <95%; however, saturations between 85 and 89% seem to increase mortality. The optimal oxygen saturation target for these infants postnatally is still unknown.
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http://dx.doi.org/10.1159/000337345DOI Listing
January 2013

Resuscitation with supplementary oxygen induces oxidative injury in the cerebral cortex.

Free Radic Biol Med 2012 Sep 24;53(5):1061-7. Epub 2012 Jul 24.

Department of Pediatric Research, University of Oslo, Oslo University Hospital, Rikshospitalet, N-0424 Oslo, Norway.

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.07.022DOI Listing
September 2012

Risks and benefits of oxygen in the delivery room.

J Matern Fetal Neonatal Med 2012 Apr 13;25 Suppl 1:41-4. Epub 2012 Mar 13.

Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy.

Oxygen is an essential element of aerobic life, and oxidative metabolism represents a principal source of energy. Nevertheless, oxygen may also be toxic and mutagenic with the potential to cause damage through the production of reactive oxygen species (ROS). ROS generation can be considered a double-edged sword. Beneficial effects of ROS occur at moderate concentrations and involve physiological roles in cellular responses to noxia, as in defense against infectious agents, in the function of a number of cellular signaling pathways and the induction of a mitogenic response. The overproduction of ROS and the insufficiency of an antioxidant mechanism results in oxidative stress (OS), a deleterious process and important mediator of damage to cell structures and tissues. Newborns, especially if preterm, are particularly susceptible to OS and damage due to increased generation of ROS, the lack of adequate antioxidant protection, and the inability to induce antioxidant defenses during the hyperoxic challenge at birth. Hence the "Oxygen Paradox": higher eukaryotic aerobic organisms cannot exist without oxygen and without OS, yet oxygen and ROS are dangerous to their existence. Originally, the oxygen paradox described that the injury was aggravated by giving oxygen after hypoxia. Today, we know this is caused by production of oxygen radicals. Therefore, it is mandatory in the handling of newborns to use oxygen as a medication when clinical surveillance indicates a need.
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http://dx.doi.org/10.3109/14767058.2012.665236DOI Listing
April 2012

Resuscitation of newborn piglets. short-term influence of FiO2 on matrix metalloproteinases, caspase-3 and BDNF.

PLoS One 2010 Dec 9;5(12):e14261. Epub 2010 Dec 9.

Department of Paediatric Research, University of Oslo and Oslo University Hospital, Rikshospitalet, Oslo, Norway.

Background: Perinatal hypoxia-ischemia is a major cause of mortality and cerebral morbidity, and using oxygen during newborn resuscitation may further harm the brain. The aim was to examine how supplementary oxygen used for newborn resuscitation would influence early brain tissue injury, cell death and repair processes and the regulation of genes related to apoptosis, neurodegeneration and neuroprotection.

Methods And Findings: Anesthetized newborn piglets were subjected to global hypoxia and then randomly assigned to resuscitation with 21%, 40% or 100% O(2) for 30 min and followed for 9 h. An additional group received 100% O(2) for 30 min without preceding hypoxia. The left hemisphere was used for histopathology and immunohistochemistry and the right hemisphere was used for in situ zymography in the corpus striatum; gene expression and the activity of various relevant biofactors were measured in the frontal cortex. There was an increase in the net matrix metalloproteinase gelatinolytic activity in the corpus striatum from piglets resuscitated with 100% oxygen vs. 21%. Hematoxylin-eosin (HE) staining revealed no significant changes. Nine hours after oxygen-assisted resuscitation, caspase-3 expression and activity was increased by 30-40% in the 100% O(2) group (n = 9/10) vs. the 21% O(2) group (n = 10; p<0.04), whereas brain-derived neurotrophic factor (BDNF) activity was decreased by 65% p<0.03.

Conclusions: The use of 100% oxygen for resuscitation resulted in increased potentially harmful proteolytic activities and attenuated BDNF activity when compared with 21%. Although there were no significant changes in short term cell loss, hyperoxia seems to cause an early imbalance between neuroprotective and neurotoxic mechanisms that might compromise the final pathological outcome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014261PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3000320PMC
December 2010

Metabolomic analyses of plasma reveals new insights into asphyxia and resuscitation in pigs.

PLoS One 2010 Mar 9;5(3):e9606. Epub 2010 Mar 9.

Department of Pediatric Research, Oslo University Hospital, Rikshospitalet, University of Oslo, Norway.

Background: Currently, a limited range of biochemical tests for hypoxia are in clinical use. Early diagnostic and functional biomarkers that mirror cellular metabolism and recovery during resuscitation are lacking. We hypothesized that the quantification of metabolites after hypoxia and resuscitation would enable the detection of markers of hypoxia as well as markers enabling the monitoring and evaluation of resuscitation strategies.

Methods And Findings: Hypoxemia of different durations was induced in newborn piglets before randomization for resuscitation with 21% or 100% oxygen for 15 min or prolonged hyperoxia. Metabolites were measured in plasma taken before and after hypoxia as well as after resuscitation. Lactate, pH and base deficit did not correlate with the duration of hypoxia. In contrast to these, we detected the ratios of alanine to branched chained amino acids (Ala/BCAA; R(2).adj = 0.58, q-value<0.001) and of glycine to BCAA (Gly/BCAA; R(2).adj = 0.45, q-value<0.005), which were highly correlated with the duration of hypoxia. Combinations of metabolites and ratios increased the correlation to R(2)adjust = 0.92. Reoxygenation with 100% oxygen delayed cellular metabolic recovery. Reoxygenation with different concentrations of oxygen reduced lactate levels to a similar extent. In contrast, metabolites of the Krebs cycle (which is directly linked to mitochondrial function) including alpha keto-glutarate, succinate and fumarate were significantly reduced at different rates depending on the resuscitation, showing a delay in recovery in the 100% reoxygenation groups. Additional metabolites showing different responses to reoxygenation include oxysterols and acylcarnitines (n = 8-11, q<0.001).

Conclusions: This study provides a novel strategy and set of biomarkers. It provides biochemical in vivo data that resuscitation with 100% oxygen delays cellular recovery. In addition, the oxysterol increase raises concerns about the safety of 100% O(2) resuscitation. Our biomarkers can be used in a broad clinical setting for evaluation or the prediction of damage in conditions associated with low tissue oxygenation in both infancy and adulthood. These findings have to be validated in human trials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009606PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834759PMC
March 2010

Resuscitation of hypoxic newborn piglets with supplementary oxygen induces dose-dependent increase in matrix metalloproteinase-activity and down-regulates vital genes.

Pediatr Res 2010 Mar;67(3):250-6

Department of Paediatric Research, University of Oslo, Oslo University Hospital, Rikshospitalet, Oslo, Norway.

The optimal oxygen concentration for newborn resuscitation is still discussed. Oxygen administration during reoxygenation may induce short- and long-term pathologic changes via oxidative stress and has been associated to later childhood cancer. The aim was to study changes in oxidative stress-associated markers in liver and lung tissue of newborn pigs after acute hypoxia followed by reoxygenation for 30 min with 21, 40, or 100% oxygen compared with room air or to ventilation with 100% oxygen without preceding hypoxia. Nine hours after resuscitation, we found a dose-dependent increase in the matrix metalloproteinase gelatinase activity in liver tissue related to percentage oxygen supply by resuscitation (100% versus 21%; p = 0.002) pointing at more extensive tissue damage. Receiving 100% oxygen for 30 min without preceding hypoxia decreased the expression of VEGFR2 and TGFBR3 mRNA in liver tissue, but not in lung tissue. MMP-, VEGF-, and TGFbeta-superfamily are vital for the development, growth, and functional integrity of most tissues and our data rise concern about both short- and long-term consequences of even a brief hyperoxic exposure.
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http://dx.doi.org/10.1203/PDR.0b013e3181cde843DOI Listing
March 2010

Newborn piglets exposed to hypoxia after nicotine or saline pretreatment: long-term effects on brain and heart.

J Matern Fetal Neonatal Med 2009 Feb;22(2):161-8

Medical Faculty, Department of Pediatric Research, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway.

Objective: We wished to assess the effect of global hypoxia and the effect of nicotine pretreatment on the brain and heart of newborn pigs. Hypothesising that nicotine might give a better outcome because of its anti-apoptotic and anti-inflammatory effects.

Methods: Twenty-two anaesthetised piglets were randomised to pretreatment with saline or nicotine (130 microg/kg/h) before 45 min global hypoxia. They were observed for 27 h. The brain and heart were assessed with histopathological methods. Serum for Troponin t (TnT) analyses was collected at baseline and at the end of the experiment.

Results: There were no significant differences between the groups. At the end of hypoxia, BE was -14.8 +/- 4.9 mmol/l and MABP was 25 +/- 9 mmHg. Seven animals had autolysis of the cerebrum/cerebellum, their BE after hypoxia was -19 +/- 1.8 mmol/l and MABP 23 +/- 3 mmHg. The remaining 15 animals had a BE of -13 +/- 4.7mmol/l (p = 0.0004) and a MABP of 26 +/- 11 mmHg (ns). Eleven animals presented myocardial damage. A significant increase in TnT occurred in both groups. TnT increase and myocardial damage correlated (p = 0.001; r = 0.67). Animals with severe increase in TnT presented severe brain damage.

Conclusions: Severe increase in serum TnT levels was linked to severe cerebral damage. Nicotine pretreatment had no impact on cerebral or cardiac histopathology.
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http://dx.doi.org/10.1080/14767050802663186DOI Listing
February 2009

Improved cognitive development among preterm infants attributable to early supplementation of human milk with docosahexaenoic acid and arachidonic acid.

Pediatrics 2008 Jun;121(6):1137-45

Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Norway.

Objective: The objective of our study was to evaluate the effect of supplementation with docosahexaenoic acid and arachidonic acid for human milk-fed preterm infants. The primary end point was cognitive development at 6 months of age.

Methods: The study was a randomized, double-blind, placebo-controlled study among 141 infants with birth weights of <1500 g. The intervention with 32 mg of docosahexaenoic acid and 31 mg of arachidonic acid per 100 mL of human milk started 1 week after birth and lasted until discharge from the hospital (on average, 9 weeks). Cognitive development was evaluated at 6 months of age by using the Ages and Stages Questionnaire and event-related potentials, a measure of brain correlates related to recognition memory.

Results: There was no difference in adverse events or growth between the 2 groups. At the 6-month follow-up evaluation, the intervention group performed better on the problem-solving subscore, compared with the control group (53.4 vs 49.5 points). There was also a nonsignificant higher total score (221 vs 215 points). The event-related potential data revealed that infants in the intervention group had significantly lower responses after the standard image, compared with the control group (8.6 vs 13.2). There was no difference in responses to novel images.

Conclusions: Supplementation with docosahexaenoic acid and arachidonic acid for very preterm infants fed human milk in the early neonatal period was associated with better recognition memory and higher problem-solving scores at 6 months.
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http://dx.doi.org/10.1542/peds.2007-1511DOI Listing
June 2008

[Dialysis treatment of infants with end stage failure].

Tidsskr Nor Laegeforen 2007 Nov;127(23):3069-72

Det medisinske fakultet, Universitet i Oslo.

Background: End stage renal failure in infants is rare, and was until recently regarded as untreatable. Advancements in dialysis techniques and other renal replacement therapy, have now made lifesaving treatment possible.

Material And Methods: Three infants who developed end stage renal failure shortly after birth and were subsequently treated with long-term dialysis (as a bridge to transplantation) are presented and their results are compared with those from other dialysis centres.

Results And Interpretation: All three patients were successfully dialysed until transplantation, two with peritoneal dialysis and one with haemodialysis. Complications were rare and manageable. The results are in accordance with findings from previous studies. Long-term dialysis in infants with chronic renal failure should no longer be considered experimental and is now a real alternative until the child is big enough to have a transplantation. Treatment outcome is affected by co-morbidity. The treatment requires a multidisciplinary approach with specialists from many fields including paediatrics, paediatric surgery, nephrology, nutrition and dialysis. In addition it is essential to cooperate with the parents, as the treatment is demanding for the family as well as for the medical personnel.
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November 2007

Resuscitation of hypoxic newborn piglets with oxygen induces a dose-dependent increase in markers of oxidation.

Pediatr Res 2007 Nov;62(5):559-63

Department of Pediatric Research, Medical Faculty, University of Oslo Rikshospitalet Medical Centre, Oslo, N-0027, Norway.

Newborn resuscitation with pure oxygen may be associated with long-term detrimental effects. Due to the change in attitude toward use of less oxygen upon resuscitation, there is a need to study effects of intermediate hyperoxia. The aim was to study dose-response correlation between inspiratory fraction of oxygen used for resuscitation and urinary markers of oxidative damage to DNA and amino acids. Hypoxemia was induced in newborn piglets following a standardized model; they were resuscitated for 15 min with either 21%, 40%, 60% or 100% oxygen and observed for 1 h. Urine samples were collected. Urinary elimination of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), 2'deoxyguanosine (2dG), ortho-tyrosine (o-Tyr) and phenylalanine (Phe) were determined by HPLC and tandem mass spectrometry (HPLC-MS/MS). Quotient of 8-oxo-dG/2dG and o-Tyr/Phe ratios were significantly and dose-dependant higher in piglets resuscitated with supplementary oxygen. 8-oxodG/dG: Mean (SD) 5.76 (1.81) versus 22.44 (12.55) p < 0.01 and o-Tyr/Phe: 19.07 (10.7) versus 148.7 (59.8)for 21% versus 100%, p < 0.001. Hypoxia and subsequent resuscitation for 15 min with graded inspiratory fraction of oxygen causes increased oxidative stress and a dose-dependant oxidation of DNA and Phenylalanine. The increase in the hydroxyl attack may lead to a pro-oxidative status and risk for genetic instability.
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http://dx.doi.org/10.1203/PDR.0b013e318156e8aaDOI Listing
November 2007