increased expression of inflammatory genes in the neonatal mouse brain after hyperoxic reoxygenation

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10.1038/pr.2014.193
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10.1038/pr.2014.193

nature publishing group Articles Translational Investigation increased expression of inflammatory genes in the neonatal mouse brain after hyperoxic reoxygenation Anne Gro W. Rognlien1, Embjørg J. Wollen1, Monica Atneosen-Åsegg1,2 and Ola Didrik Saugstad1 Background: Hyperoxic reoxygenation following hypoxia increases the expression of inflammatory genes in the neona tal mouse brain. We have therefore compared the temporal profile of 44 a priori selected genes after hypoxia and hyperoxic or normoxic reoxygenation. Methods: Postnatal day 7 mice were subjected to 2 h of hypoxia (8% O2 ) and 30 min reoxygenation with 60% or 21% O2 . After 0 to 72 h observation, mRNA and protein were exam ined in the hippocampus and striatum. results: There were significantly higher gene expression changes in six genes after hyperoxic compared to normoxic reoxygenation. Three genes had a generally higher expression throughout the observation period: the inflammatory genes Hmox1 (mean difference: 0.52, 95% confidence interval (CI): 0.15–1.01) and Tgfb1 (mean difference: 0.099, CI: 0.003–0.194), and the transcription factor Nfkb1 (mean difference: 0.049, CI: 0.011–0.087). The inflammatory genes Cxcl10 and Il1b, and the DNA repair gene Neil3, had a higher gene expression change after hyperoxic reoxygenation at one time point only. Nineteen genes involved in inflammation, transcription regulation, apoptosis, angiogenesis, and glucose transport had signifi cantly different gene expression changes with time in all inter vention animals. conclusion: We confirm that hyperoxic reoxygenation induces a stronger inflammatory gene response than reoxy genation with air. neonatal asphyxia is still one of the leading causes of mortality in children under 5 y of age worldwide (1). Hypoxia–reoxygenation is an important mechanism involved in the pathogenesis of injury caused by neonatal asphyxia (2,3). Hypoxia–reoxygenation leads to inflammation and augmented oxidative stress through energy failure, increased production of reactive oxygen and nitrogen species, and decreased antioxi dant activity. This may subsequently induce lipid peroxidation, increased cellular permeability, glutamate excitotoxicity, and damage to proteins and DNA, ultimately resulting in necrosis and apoptosis (3–5).The neonatal brain is particularly vulner able to oxidative stress (5,6). Until recently, hyperoxic reoxygenation was recommended when initiating neonatal resuscitation (7). However, many studies on hyperoxic reoxygenation after asphyxia, hypoxia ischemia (HI), and hypoxia have been conducted both in humans and animals (reviewed in refs. (8,9)), concluding that substantial evidence supports that starting with air is better than 100% O2 when resuscitating term neonates, and hyper oxia should be avoided (9). Hyperoxia alone has also been shown to have detrimental effects (10). However, there is still insufficient evidence to give recommendations on which strat egy of supplemental oxygen to use in moderately and late pre mature neonates (7). Although meta-analyses show that resuscitation with air after neonatal asphyxia in term neonates gives lower mortality than resuscitation with 100% O2 (11), the mechanisms have not been fully elucidated. One avenue of research is gene expres sion profiling. Hedtjärn et al. (12,13) have studied gene expres sion profiling after HI in neonatal mice. However, to the best of our knowledge, no other groups have studied gene expression profiling after hyperoxic reoxygenation. We have established a model subjecting postnatal day seven (P7) mice to a global hypoxic event followed by reoxygenation with different frac tions of inspired oxygen (14). P7 mice have a brain develop ment comparable to moderate and late premature humans at GA 32–36 wk (15). Moderate and late preterm neonates have a higher risk of neonatal mortality and morbidity and also a higher risk of later sequelae than term neonates, and there has been a call for research into this large group of neonates (16). In a recent study from our group, we presented the transcrip tome profile in the forebrain of P7 mice 2.5 h after hypoxia and reoxygenation (17). Hyperoxic reoxygenation gave increased expression of inflammatory genes and decreased expression of genes of the oxidative phosphorylation pathway. To explore further the gene expression changes of the brain after hypoxia and hyperoxic reoxygenation, we made a tempo ral profile of genes in the hippocampus and striatum. Hazelton et al. (18) found increased inflammation and neuronal cell death in the hippocampus, and decreased performance on the Morris Water Maze, after hyperoxic reoxygenation in adult rats. Problems with cognition and memory are also common among The first two authors contributed equally to this work. 1Department of Pediatric Research, University of Oslo, Oslo University Hospital HF, Oslo, Norway; 2Department of Clinical Molecular Biology and Laboratory Sciences, Akershus University Hospital, Lørenskog, Norway. Correspondence: Anne Gro W. Rognlien (agwlien@rr-research.no) Received 25 April 2014; accepted 5 September 2014; advance online publication 24 December 2014. doi:10.1038/pr.2014.193 326 Pediatric ReSeARCH
Copyright © 2015 International Pediatric Research Foundation, Inc. Volume 77 | Number 2 | February 2015 Hyperoxic reoxygenation and inflammation
Articles moderate and late preterm neonates (16). The striatum is one of the first regions to show damage on magnetic resonance imaging after mild to moderate asphyxia (19). In our transcrip tome profiling study, there were many differentially expressed genes after reoxygenation with 60% O2 (17). 100% O2 is less clinically relevant after the International Liaison Committee on Resuscitation guidelines changed in 2010 (7); thus we decided on 60% O2 as hyperoxia. Forty-four target genes were selected (Table 1) based on our transcriptome profiling studies (14,17), a pilot study and literature search. The objective of the pres ent study was to compare the gene expression changes in the hippocampus and striatum of P7 mice after hypoxia followed by reoxygenation with 60% O2 or air (21% O2 ), and to make a temporal profile of the genes up to 3 d after the intervention. RESULTS P7 mice were randomized to hypoxia in 8% O2 for 2 h, followed by reoxygenation for 30 min with either 60% O2 (H60) or air (H21), or controls (C) kept in air for the duration of the experi ment (Figure 1). mRNA and protein were examined in hip pocampus and striatum. There were no statistical differences between main groups in weight or sex distribution in either the mRNA or protein samples (data not shown). Blood glucose was significantly lower in the period of sepa ration from the dams in both hypoxia and control mice com pared to baseline (Supplementary Figure S1 online). Blood glucose was also significantly lower in the hypoxia mice com pared to control mice right after hypoxia and at 0 h after reoxy genation, but at 2.5 h after reoxygenation, the blood glucose of hypoxia mice had returned to the level of controls. Gene Expression After Hypoxia–Reoxygenation There was a statistically higher gene expression change in six genes related to inflammation, transcription regulation, and DNA repair after reoxygenation with 60% O2 (H60) compared to air (H21) (Figure 2). Three genes had an overall higher expression in the H60 group in the general linear model: The inflammatory genes Hmox1 (for gene name see Table 1, H60 vs. H21 mean difference: 0.52, 95% confidence interval (CI): 0.15–1.01, P = 0.009) and Tgfb1 (mean difference: 0.099, CI: 0.003–0.194, P = 0.044), and the transcription factor Nfkb1 (mean difference: 0.049, CI: 0.011–0.087, P = 0.012). At single time points, we found a higher gene expression change after hyperoxic reoxygenation at 0 h of the inflammatory gene Cxcl10 (Mann–Whitney U-test, P = 0.009) and the DNA repair gene Neil3 (mean difference: 0.17, CI: 0.001–0.34, P = 0.048), and at 5 h of the inflammatory gene Il1b (Mann Whitney U-test, P = 0.021). Patterns of Gene Expression Temporal Profiles Nineteen genes involved in inflammation, transcription regu lation, apoptosis, angiogenesis, and glucose transport, had significantly different gene expression changes with time after hypoxia and reoxygenation in the general linear model in all intervention animals compared to controls. We identified three patterns of gene expression temporal profiles: (i) Genes that were highly increased in expression right after reoxygen ation at 0 h, and then decreased (Bnip3, Ccl2, Edn1, Il6, Mt1, Slc2a1, Vegfa, gene names and main functions in Table 1) (Figures 3 and 4a); (ii) Genes that had the highest increase in expression between 2.5 and 5 h after completed reoxygenation (Ccl12, Ccl5, Cxcl10, Hmox1, Il1b, Lcn2, Stat3, Tnf) (Figures 2, 3, and 4b); (iii) Genes where the expression fluctuated around fold change of 1, but had significant differences between time points (Apaf1, Ccnd1, Neil3, and Nfkb1) (Figures 2 and 4c). Protein Analysis For seven of the genes with significant changes in mRNA expression, protein concentration was measured by enzyme linked immunosorbent assay in tissue homogenate from the hippocampus and striatum (Gene symbolb and gene names in Table 1). The proinflammatory chemokine CCL12 had peak mRNA expression after 2.5 h, while the peak protein concentra tion was after 12 h (Figure 3). CCL12 had a higher concentration in the H60 group than the H21 group in the general linear model (mean difference 1.2 pg/mg protein, CI: 0.11–2.3, P = 0.032), although no such difference could be detected in mRNA after hyperoxic reoxygenation. The increased CCL12 concentration after hyperoxic reoxygenation started already at 0 h after reoxy genation, before the mRNA peak. Vascular endothelial growth factor A, important in angiogenesis, had a significant increase in protein concentration in all intervention animals compared to controls at 0 h (mean difference: H21-C 44.3 pg/mg prot, CI: 28.5–60.1; mean difference: H60-C 49.4, CI: 33.9–64.9), concur ring with the peak mRNA expression. LCN2 is an iron chelator involved in the innate immune and stress response. It showed a significant change in protein concentration with time in the hypoxia groups with a peak expression after 12 h, but there were no significant differences between hypoxia and controls at any time points. Four proteins had no significant differences at the time points analyzed: IL1B, MT1, NEIL3, and STAT3. DISCUSSION In this study, we have used a P7 mouse model of hypoxia fol lowed by reoxygenation with 60% O2 or air, to examine the gene expression changes of 44 a priori selected genes in the hippocampus and striatum, and to make a temporal profile up to 3 d after the intervention. Our main result is that hyper oxic reoxygenation gave a significantly higher gene expression change in six genes involved in inflammation (Cxcl10, Il1b, Tgfb1, Hmox1), transcription regulation (NfKb1), and DNA repair (Neil3). The augmented inflammatory gene response was still evident at 72 h after completed reoxygenation. Other studies have also shown increased inflammatory and oxidative stress response after hyperoxic reoxygenation, although, most have used 100% O2 in either term or immature animal models, while in this study we have used 60% O2 for 30 min in a moder ate to late preterm model. In our transcriptome profiling study, we have recently shown that hyperoxic reoxygenation induced increased expression of inflammatory genes in the neonatal mice brain (17). Previously, we have reported a dose dependent increase in markers of Pediatric ReSeARCH 327 Copyright © 2015 International Pediatric Research Foundation, Inc. Volume 77 | Number 2 | February 2015
Copyright © 2015 International Pediatric Research Foundation, Inc. Articles Rognlien et al. table 1. Target and reference genes Gene symbol Gene name Main function(s) Apaf1a Apoptotic peptidase activating factor 1 Apoptosis Apex1 Apurinic/apyrimidinic endonuclease 1 DNA repair, oxidative stress Atm Ataxia telangiectasia mutated homolog (human) Cell cycle arrest Bax BCL2-associated X protein Apoptosis Bcl2 B cell leukemia/lymphoma 2 Apoptosis Bcl2l1 BCL2-like 1 Apoptosis Bnip3 BCL2/adenovirus E1B interacting protein 3 Autophagy Casp3 Caspase3 Apoptosis Ccl12b Chemokine (C-C motif) ligand 12 Inflammation Ccl2b Chemokine (C-C motif) ligand 2 Inflammation Ccl5 Chemokine (C-C motif) ligand 5 Inflammation Ccnb1 Cyclin B1 Cell cycle arrest Ccnd1 Cyclin D1 Cell cycle arrest Chek1 Checkpoint kinase 1 Cell cycle arrest Cxcl10b Chemokine (C-X-C motif) ligand 10 Inflammation Edn1 Endothelin 1 Angiogenesis Fasl fas ligand (TNF superfamily, member 6) Apoptosis Gadd45g Growth arrest and DNA-damage inducible 45γ Cell cycle arrest Grin2b Glutamate receptor, ionotropic, NMDA2B (epsilon 2) Oxidative stress Hif1a Hypoxia inducible factor 1, α-subunit Response to hypoxia, transcription factor Hmox1 Heme oxygenase (decycling) 1 Inflammation, oxidative stress Igf1r Insulin-like growth factor I receptor Glucose regulation Ikbkb Inhibitor of κB kinase-β Apoptosis Il10 Interleukin-10 Inflammation Il1bb Interleukin-1β Inflammation, apoptosis Il6 Interleukin-6 Inflammation Jun jun proto-oncogene Transcription factor Lcn2b Lipocalin 2 Inflammation Mapk14 Mitogen-activated protein kinase 14 Inflammation Mt1b Metallothionein 1 Oxidative stress Mutyh mutY homolog (Escherichia coli) DNA repair Neil1 nei endonuclease VIII-like 1 (E. coli) DNA repair Neil3b nei like 3 (E. coli) DNA repair Nfkb1 Nuclear factor of κ light polypeptide gene enhancer in B cells 1, p105 Transcription factor, inflammation Ogg1 8-oxoguanine DNA-glycosylase 1 DNA repair Prkaa1 Protein kinase, AMP-activated, α1 catalytic subunit Glucose regulation Slc2a1 Solute carrier family 2 (facilitated glucose transporter), member 1 Glucose transport Sod1 Superoxide dismutase 1, soluble Oxidative stress Stat3b Signal transducer and activator of transcription 3 Transcription factor Tgfb1 Transforming growth factor, β1 Inflammation, growth factor Tlr4 Toll-like receptor 4 Inflammation Tnfb Tumor necrosis factor Apoptosis, inflammation Trp53 Transformation related protein 53 Transcription factor Vegfab Vascular endothelial growth factor A Angiogenesis Actb Actin, β Reference gene, lung Polr2a Polymerase (RNA) II (DNA directed) polypeptide A Reference gene, brain and lung Ubc Ubiquitin C Reference gene, brain 18S Manufacturer control aBold: Genes mentioned in the manuscript. bAlso attempted analyzed for protein. 328 Pediatric ReSeARCH Volume 77 | Number 2 | February 2015 Hyperoxic reoxygenation and inflammation
Articles Hypoxia 8% O2 2 h Mice P7 Reox air 30 min H21 Reox 60% O2 30 min H60 Control air, C After hypoxia Observation 0 h to 72 h Baseline 0 h 2.5 h 5 h 12 h 24 h 72 h Protein Glucose Glucose mRNA Protein Glucose mRNA Glucose mRNA Glucose Protein P rotein mRNA Protein Glucose Figure 1. Experimental design. P7 mice were randomized to either hypoxia for 2 h or controls kept in air. The hypoxia group was further randomized to 30 min reoxygenation with either 60% O2 (H60) or air (H21). The hippocampus and striatum were harvested right after reoxygenation (0 h), or 2.5, 5, or 72 h after reoxygenation in the mRNA experiment (n = 21 at each time point); at baseline and 0, 12, 24, or 72 h after reoxygenation in the protein experiments (n = 7, 22, 21, 21, 21, respectively); and blood glucose was measured at baseline, after hypoxia, and 0, 2.5, 5, or 72 h after reoxygenation (n = 12, 17, 48, 39, 16, 13, respectively). a RQ (median, IQR) b 14 12 10 0 2 4 6 8 * 5 4 3 2 1 oxidative stress with increasing oxygen concentration during reoxygenation in neonatal pigs (20). In NT2-N neurons Almaas et al. (21) found increased CXCL10 after oxidative stress, and we have earlier reported increased CXCL10 after hypoxia, but with no augmented response after hyperoxic reoxygenation (22). In concordance with our study, increased expression of Il1b has been found after hyperoxic reoxygenation in fetal sheep (23), and after hyperoxia alone in neonatal rats (10). Perez-Polo and coworkers found a stronger inflammatory response after hyper oxic reoxygenation following HI in rats (24,25). We also found increased protein concentration of CCL12, a proinflammatory chemokine, after hyperoxic reoxygenation, consistent with our recent reports of increased Ccl12 expression in the eye and brain of neonatal mice after hyperoxic reoxygenation (17,26). Mojsilovic-Petrovic et al. (27) found increased expression of Ccl12 in astrocyte cultures from P7 mice after hypoxia alone. Hmox1 expression is responsive to both hypoxia and hyper oxia (28), and we found that hyperoxic reoxygenation gave a stronger induction of Hmox1 gene expression than reoxygen ation with air. In contrast to our findings, Bergeron et al. (29) only found increased expression of Hmox1 after HI in neonatal c † 0 1 2 3 4 5 d RQ (mean, CI) 1.6 1.4 1.2 1.0 * 72 0.8 0.6 0.4 0.2 0.0 0 1 2 3 4 5 72 0 e 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0 1 2 3 4 5 † 72 72 f 5 4 3 2 1 0 1.6 1.4 1.2 1.0 0.8 0.6 * 0 1 2 3 4 5 † 72 0 1 2 3 4 5 0 1 2 3 4 5 7 2 Hours after intervention Hours after intervention Hours after intervention Figure 2. Genes with significantly higher expression after hyperoxic reoxygenation. Gene expression change, relative quantification (fold change com pared to controls), of animals subjected to hypoxia and reoxygenation, and then observed for 0, 2.5, 5, or 72 h after the intervention. In six genes, there was significantly higher expression after hyperoxic than normoxic reoxygenation either overall in a general linear model or at a single time point. Data expressed as mean, confidence interval, or median, interquartile range (IQR), depending on the distribution of the data. Panel a, Cxcl10; panel b, Hmox1; panel c, Il1b; panel d, Neil3; panel e, Nfkb1; panel f, Tgfb1. Gene names are shown in Table 1. Solid line: Group subjected to hypoxia and reoxygenation with 60% O2 (H60). Dashed line: Group subjected to hypoxia and reoxygenation with air (H21). Dotted line: Average fold change of 1 for controls. *P < 0.05 H60-H21 at a single time point. †P < 0.05 H60-H21 across all time points in the general linear model. Copyright © 2015 International Pediatric Research Foundation, Inc. Pediatric ReSeARCH 329 Volume 77 | Number 2 | February 2015
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Rognlien et al. b a mRNA RQ 20 Protein concentration 15 10 c 0 1 2 3 4 5 0 5

0 6 12 † 18 24 72 0 6 12 18 Hours after intervention 24 72 20 15 10 0 5 600 500 400 300 200 100 0

0 6 12 18 24 72 0 6 12 18 Hours after intervention 24 72 120 100

0 1 2 3 4 80 60 40 20 0 0 6 12 18 24 * 72 0 6 12 18 24 72 Hours after intervention Figure 3. Genes with significant changes in both mRNA expression and protein concentration. mRNA expression (relative quantification, fold change compared to controls, median, IQR, top three panels) and protein concentration (protein concentration, pg/mg protein, bottom three panels) for the same gene with time after hypoxia and reoxygenation. Panel a: CCL12 had an overall higher protein concentration after hyperoxic reoxygenation. The peak mRNA expression was after 2.5 h and the peak protein concentration was after 12 h. Panel b: LCN2 had significant differences with time in both pro tein concentration and gene expression. Panel c: vascular endothelial growth factor A had peak both protein concentration and mRNA expression right after reoxygenation at 0 h. Gene names are shown in Table 1. Observation time after the intervention: mRNA 0, 2.5, 5, or 72 h; protein 0, 12, 24, or 72 h. Solid line: Group subjected to hypoxia and reoxygenation with 60% O2 (H60). Dashed line: Group subjected to hypoxia and reoxygenation with air (H21). Dotted line: Controls kept in air, average fold change of 1 in mRNA group. †p < 0.05 H60-H21 in the general linear model; *p < 0.05 compared to controls. rats, but not after hypoxia alone. The growth factor Tgfb1 is known to be involved in hyperoxia-induced injury in the lung (30), but to our knowledge, increased expression has not been reported earlier in relation to hyperoxia in the brain. Hyperoxic reoxygenation also gave increased expression of the pluripotent transcription factor Nfkb1,which is known to be involved in hypoxia–reoxygenation injury (3). We have ear lier reported increased Nfkb1 after hyperoxic reoxygenation in P5 mice (31), in concordance with the present study. The DNA glycosylase Neil3 is important in the repair of oxidative DNA-damage. Dalen et al. (32) reported decreased expression of Neil3 in neonatal pigs after post hypoxic hypothermia, but with no statistically significant difference between hyperoxic or normoxic reoxygenation. In this study, Neil3 was signifi cantly less suppressed in animals reoxygenated with 60% O2

compared to air, possibly suggesting increased need of DNA repair after hyperoxic reoxygenation. We identified three patterns of gene expression temporal pro files in intervention animals compared to controls: Firstly, high expression right after the intervention and rapidly decreasing; secondly, highest expression from 2.5–5 h after the interven tion; and thirdly, fluctating expression with fold change around one. Among genes induced within 5 h after the intervention, there were several genes involved in inflammation and oxida tive stress (Ccl12,Ccl2, Ccl5, Cxcl10, Hmox1, Il1b, Il6, Lcn2, Mt1, Stat3, Tnf), and this study confirms that hypoxia–reox ygenation induces inflammatory and oxidative stress respon sive genes. Other groups have found an increased expression of inflammatory genes after HI (13) and hypoxia alone (33). Genes involved in angiogenesis (Edn1, Vegfa), and the facilita tive glucose transporter Slc2a1 (homolog to GLUT1) were also 330 Pediatric ReSeARCH
induced within 5 h after the intervention. Vegfa is known to be induced by hypoxia (34), but to our knowledge, increased expression of Edn1 has not previously been described after hypoxia–reoxygenation or HI in the brain. Upregulation of glucose transport after HI has been described previously in both brain hemispheres applying the Vannucci model (5,35). In this study, the hypoxia mice had lower blood glucose right after hypoxia–reoxygenation, and this might contribute to the induction of Slc2a1 that we found. Inflammation is considered an important cause of damage after hypoxia–reoxygenation injury (2,6,13,36,37), and there is evidence that increased inflammation aggravates such injury (36). Several groups have found increased brain lesions in neo natal rats after hyperoxic reoxygenation following HI (25,38,39). The increased inflammatory gene response after hyperoxic reox ygenation we found in the present study, supports the evidence that increased inflammation might be one of the contributing factors to the increased brain damage after hyperoxic reoxygen ation. In our previous transcriptome profiling study, we found that hypoxia–reoxygenation led to increased microglial activa tion, as a sign of increased inflammation, but with no differ ence between normoxic and hyperoxic reoxygenation (17), in contrast to the findings of Hazelton et al. (18). Large treatment groups would probably be needed to show the subtle patho logical differences expected when comparing groups where the only difference is 30 min air or 60% O2 during reoxygenation. However, it has been hypothesized that there is a continuous specter of insults and outcomes, thus small differences in an insult may give differences in the outcome (40). Some limitations of this study are that only 44 target genes were examined, and not the whole genome, and there was Copyright © 2015 International Pediatric Research Foundation, Inc. Volume 77 | Number 2 | February 2015 a Hyperoxic reoxygenation and inflammation
Articles 2.0 RQ (mean, CI) RQ (median, IQR) b RQ (mean, CI) c RQ (mean, CI) 1.5 1.0 0.5 0.0 2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 1.2 1.1 1.0 0.9 0.8 0.7 Bnip3 72 10.0 RQ (median, IQR) 8.0 6.0 4.0 2.0 0.0 Ccl2 6.0 4.0 2.0 RQ (median, IQR) 0.0 Edn1 0 1 2 3 4 5 6 7 2 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 2 Il6 0 1 2 3 4 5 6 Hours after intervention Stat3 72 0 1 2 3 4 5 6 Hours after intervention Apaf1 72 0 1 2 3 4 5 6 Hours after intervention 72 3.0 2.0 1.0 RQ (median, IQR) RQ (median, IQR) RQ (mean, CI) 0.0 3.0 2.0 1.0 0.0 1.4 1.2 1.0 0.8 0.6 Mt1 0 1 2 3 4 5 6 Hours after intervention Ccl5 72 0 1 2 3 4 5 6 Hours after intervention Ccnd1 72 0 1 2 3 4 5 6 Hours after intervention 72 4.0 3.0 2.0 RQ (median, IQR) RQ (median, IQR) 1.0 0.0 4.0 3.0 2.0 1.0 0.0 Slc2a1 0 1 2 3 4 5 6 Hours after intervention Tnf 72 0 1 2 3 4 5 6 Hours after intervention 72 Figure 4. Temporal profile patterns of gene expression changes. We identified three temporal patterns of gene expression changes after hypoxia and reoxygenation. Panel a: Genes that were highest in expression right after reoxygenation and then decreased. Panel b: Genes that had the highest expres sion between 2.5 and 5 h after the intervention. Panel c: Genes with fold change fluctuating around 1. Data expressed as relative quantification, fold change compared to controls, in mean, confidence interval, or median, interquartile range (IQR), depending on the distribution of the data. Gene names are shown in Table 1. Solid line: Animals given hypoxia and reoxygenation with 60% O2 (H60). Dashed line: Animals given hypoxia and reoxygenation with air (H21). Dotted line: Control animals with average fold change of 1. unfortunately not enough tissue to study both the mRNA expression and protein concentration in each mouse. Neither was there enough tissue to analyze the protein concentration for all 19 genes with significant changes in mRNA. The absolute differences in gene expression, although statistically significant, are not very large, and this study cannot conclude on the bio logical significance of the observations. However, the changes in mRNA expression and protein concentrations were mainly consistent, and earlier studies have reported biologically differ ent phenotypes with a 20% change in gene expression (41). Copyright © 2015 International Pediatric Research Foundation, Inc. In conclusion, we have shown an increased inflamma tory gene response after hyperoxic reoxygenation following a hypoxic episode in neonatal mice. This could contribute to the increased morbidity and mortality after hyperoxic reoxygen ation seen in humans (11). METHODS Animals The Norwegian Animal Research Authority approved all experiments (FOTS ID 3112 and 4543), and animals were cared for and handled in accordance with Norwegian legislation and Directive 2010/63/EU. Pediatric ReSeARCH 331 Volume 77 | Number 2 | February 2015
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Rognlien et al. Protein Analysis Mice of strain C57BL/6JBomTac were bought in December 2008 (Taconic, Ejby, Denmark), and inbred locally. The mice were sta bled in ventilated Green-line cages (Techniplast, Varese, Italy) with standard bedding at 24 °C on a 12:12 h light/dark cycle with access to standard rodent pellets and water ad libitum. Dams nursed their own pups. In total, 333 pups were used in the experiments, including pilots and testing, of which 19 died during the experiment and were excluded before analysis (mortality: 5.7%). Experimental Model Mice pups on P7 of both sexes were used in the experiments (day of discovery = P1). The pups were randomized to either hypoxia in 8% O2 for 2 h or controls (C) kept in air for the duration of the experi ment. This hypoxic event has previously been shown to give the pups a respiratory compensated metabolic acidosis fulfilling the BE-criteria for neonatal asphyxia (14). The hypoxia group was randomized fur ther to reoxygenation with either 60% O2 (H60) or air (H21) (Figure 1). After the designated observation period, the mice were decapi tated and rapidly dissected on ice. The hippocampus and striatum were harvested, snap frozen in liquid nitrogen, and stored at −79 °C. For all experiments, hippocampus and striatum were pooled from the same animal and analyzed together. Blood glucose was measured (Freestyle Light, Abbott, Abbott Park, IL) right after decapitation at baseline (right after separation from the mother, n = 12), after hypoxia before reoxygenation (n = 17), right after completed reoxygenation (0 h, n = 48), or at 2.5, 5, and 72 h after completed reoxygenation (n = 39, 16, and 13, respectively) (Figure 1). Due to the small size of the animals, there was no repeated sampling. Custom made Lucite chambers with floor heating were used for the experiments. Hypoxia was induced with premixed gas 8% O2 bal anced with nitrogen (Yara, Oslo, Norway, from 2012 Aga, Munich, Germany). Reoxygenation was performed in separate chambers with either air, or 60% O2 achieved with an conventional oxygen mixer. Atmospheric pressure was maintained through a continuous flow of inlet gas of minimum 0.5 l/min and open outlets, CO2 concentra tion was less than 0.3%. An open water source kept air humidity at 40–50%. The skin temperature of the mice was kept close to nesting temperature at 34–36 °C through monitoring with IR-thermometer (Fluke 62 mini, Fluke, Everett, WA). The mice were not anesthetized or restrained. For the intervention and observation up to 2.5 h, the pups were separated from their dams, but pups with longer desig nated observation were returned. Real-Time Reverse-Transcriptase PCR Pooled samples of hippocampus and striatum from the same animal were prepared and total RNA was extracted, reverse transcribed to cDNA, and amplified as described previously (14). TaqMan array microfluidic cards (Life Technologies, Carlsbad, CA) were used to study gene expression changes. 250 ng cDNA was used in each fill reservoir and the cards were prepared and analyzed in Applied bio systems 7900HT Fast Real-time PCR system (Life Technologies) with 40 thermal cycles according to the manufacturer’s instructions. All samples were run in duplicates. Eighteen possible mouse reference genes were tested in 16 samples in triplicates (TaqMan Array Mouse Endogenous Control Panel). The results were analyzed with Qbase+ (Biogazelle, Zwijnaarde, Belgium), and Pol2ra and Ubc were cho sen as reference genes in the brain samples. In a pilot study, TaqMan Mouse immune panel was analyzed in 20 samples. For the main study, a Custom TaqMan array micro fluidic card (Life Technologies) was designed with 44 target genes suitable for either brain or lung tissue (List of assays in Supplementary Table S1 online), three reference genes, and one manufacturer control, in eight replicates on the 384 well card. Lung results will be presented in a separate article. Gene expression changes were evaluated using the comparative CT method of relative quantification (2−ΔΔCT). Eighty-four samples were analyzed with the Custom TaqMan array. The mice were sacrificed either right after completed reoxygenation (0 h), or 2.5, 5, or 72 h after completed reoxygenation (Figure 1, C n = 5, H21 n = 8, and H60 n = 8 at all time points). The 2.5 and 5 h time points were chosen to examine the gene expression changes in the window between first and second energy failure (42). PCR evaluation determined one extreme outlier in the H60 group at 5 h, which excluded this mouse from statistical analysis. 332 Pediatric ReSeARCH
The frozen hippocampus and striatum were homogenized in lysis buffer (Tris-HCL) containing 1% NP-40 and a protease inhibitor cock tail (Roche diagnostics, Basel, Switzerland) using Omni tip (OMNI International, Kennesaw, GA), and centrifuged twice at 12,000 × g, 4 °C, for 15 min. The supernatant was stored in −79 °C until analy sis. Protein concentration was measured using BioRad Protein Assay and Multiskan Ascent spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Ten proteins were chosen for analysis (Gene nameb in Table 1) based on the mRNA results. We conducted a pilot of LCN2, and chose time points of organ harvest at baseline, or at 0, 12, 24, or 72 h after completed reoxygenation (n = 7, 22, 21, 21, and 21, respectively). The commercially available enzyme-linked immu nosorbent assay kits Mouse CCL12 (MCC120, dilution 1:1), Mouse CCL2 (MJE00, dilution 1:1), Mouse CXCL10 (MCX10, dilution 1:1), Mouse IL1B (MLB00C, dilution 1:1), Mouse LCN2 (DY1857, dilu tion 1:10), Mouse TNF-α (MTA00B, dilution 1:1), and Mouse/rat vascular endothelial growth factor (MMV00, dilution 1:2) from R&D (R&D, Minneapolis, MN) and MT1 (E91119Mu, dilution 1:5), NEIL3 (E82291Mu, dilution 1:5), and STAT3 (E91743Mu, dilution 1:5) from USCN Life sciences (Wuhan, People’s Republic of China) were bought and samples analyzed according to the manufacturer’s instructions. Three proteins (TNFA, CXCL10, and CCL2) had values below mini mum when testing the enzyme-linked immunosorbent assay kits, and were not analyzed on the complete set of samples. Statistical Analysis All statistical analyzes were performed with PASW statistics 18 (IBM, Armonk, NY). A general linear model was developed with time and reoxygenation as fixed factors, and multiple comparisons were performed with Bonferroni post hoc correction. If the interaction between time and reoxygenation was not significant, the interaction was excluded from the final model. Based on line plots of the data, we determined that for some genes, analysis at each time point separately was more suitable. This method was also used if the interaction was significant in the general linear model. Single time point comparisons with three or more groups were performed with One-way ANOVA with post hoc Bonferroni correction for normally distributed data, or Kruskal–Wallis one-way ANOVA with post hoc Mann–Whitney U-test for non-normally distributed data. For two groups, single time point comparisons were performed with Student’s t-test, or Mann Whitney U-test if the data were not normally distributed. A 95% CI was chosen. We did not correct for analyzing 44 genes. Equal vari ances were assumed unless Levene’s test was significant. All graphs were made in GraphPad Prism 6 (GraphPad, La Jolla, CA). SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/pr ACKNOWLEDGMENTS The authors want to express their sincere gratitude to Grethe Dyrhaug and Maren Bakkebø for technical assistance; the staff at Comparative Medicine, Oslo University Hospital; Are Hugo Pripp for statistical assistance; and Anna Frengen, Tonje Sonerud, and the Epigen laboratory at the Department of Clinical Molecular Biology and Laboratory Sciences, Akershus University Hospital, for generously lending us equipment and their assistance in run ning TaqMan array microfluidic card. STATEMENT OF FINANCIAL SUPPORT This study was supported by Norwegian Research Council, Oslo, Norway; Laerdal Foundation, Stavanger, Norway; Renée and Bredo Grimsgaard’s Foundation, Oslo, Norway; Norwegian SIDS and Stillbirth Society, Oslo, Nor way; Kvinne-Barn Klinikkens Forskningsfond, Oslo, Norway; Tom Wilhelmsen Foundation, Oslo, Norway. REFERENCES

  1. UN Inter-agency Group for Child Mortality Estimation. Levels & Trends in Child Mortality Report 2014. http://www.unicef.org/media/files/Lev els_and_Trends_in_Child_Mortality_2014.pdf.
  2. Volpe JJ. Perinatal brain injury: from pathogenesis to neuroprotection. Ment Retard Dev Disabil Res Rev 2001;7:56–64. Volume 77 | Number 2 | February 2015 Copyright © 2015 International Pediatric Research Foundation, Inc. Hyperoxic reoxygenation and inflammation
    Articles
  3. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxy genation injury. Am J Physiol, Cell Physiol 2002;282:C227–41.
  4. Mishra OP, Delivoria-Papadopoulos M. Cellular mechanisms of hypoxic injury in the developing brain. Brain Res Bull 1999;48:233–8.
  5. McLean C, Ferriero D. Mechanisms of hypoxic-ischemic injury in the term infant. Semin Perinatol 2004;28:425–32.
  6. Vexler ZS, Tang XN, Yenari MA. Inflammation in adult and neonatal stroke. Clin Neurosci Res 2006;6:293–313.
  7. Perlman JM, Wyllie J, Kattwinkel J, et al.; Neonatal Resuscitation Chapter Collaborators. Part 11: Neonatal resuscitation: 2010 International Consen sus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations. Circulation 2010;122(16 Suppl 2):S516–38.
  8. Richmond S, Goldsmith JP. Air or 100% oxygen in neonatal resuscitation? Clin Perinatol 2006;33:11–27, v.
  9. Goldsmith JP, Kattwinkel J. The role of oxygen in the delivery room. Clin Perinatol 2012;39:803–15.
  10. Felderhoff-Mueser U, Sifringer M, Polley O, et al. Caspase-1-processed interleukins in hyperoxia-induced cell death in the developing brain. Ann Neurol 2005;57:50–9.
  11. Saugstad OD, Ramji S, Soll RF, Vento M. Resuscitation of newborn infants with 21% or 100% oxygen: an updated systematic review and meta-analy sis. Neonatology 2008;94:176–82.
  12. Hedtjärn M, Mallard C, Eklind S, Gustafson-Brywe K, Hagberg H. Global gene expression in the immature brain after hypoxia-ischemia. J Cereb Blood Flow Metab 2004;24:1317–32.
  13. Hedtjärn M, Mallard C, Hagberg H. Inflammatory gene profiling in the developing mouse brain after hypoxia-ischemia. J Cereb Blood Flow Metab 2004;24:1333–51.
  14. Wollen EJ, Sejersted Y, Wright MS, et al. Transcriptome profiling of the newborn mouse lung after hypoxia and reoxygenation: hyperoxic reoxy genation affects mTOR signaling pathway, DNA repair, and JNK-pathway regulation. Pediatr Res 2013;74:536–44.
  15. Hagberg H, Peebles D, Mallard C. Models of white matter injury: compari son of infectious, hypoxic-ischemic, and excitotoxic insults. Ment Retard Dev Disabil Res Rev 2002;8:30–8.
  16. Raju TN, Higgins RD, Stark AR, Leveno KJ. Optimizing care and outcome for late-preterm (near-term) infants: a summary of the workshop spon sored by the National Institute of Child Health and Human Development. Pediatrics 2006;118:1207–14.
  17. Wollen EJ, Sejersted Y, Wright MS, et al. Transcriptome profiling of the newborn mouse brain after hypoxia-reoxygenation: hyperoxic reoxygen ation induces inflammatory and energy failure responsive genes. Pediatr Res 2014;75:517–26.
  18. Hazelton JL, Balan I, Elmer GI, et al. Hyperoxic reperfusion after global cerebral ischemia promotes inflammation and long-term hippocampal neuronal death. J Neurotrauma 2010;27:753–62.
  19. Triulzi F, Parazzini C, Righini A. Patterns of damage in the mature neonatal brain. Pediatr Radiol 2006;36:608–20.
  20. Solberg R, Andresen JH, Escrig R, Vento M, Saugstad OD. Resuscitation of hypoxic newborn piglets with oxygen induces a dose-dependent increase in markers of oxidation. Pediatr Res 2007;62:559–63.
  21. Almaas R, Hankø E, Mollnes TE, Rootwelt T. Dexamethasone reduces bilirubin-induced toxicity and IL-8 and MCP-1 release in human NT2-N neurons. Brain Res 2012;1458:12–7.
  22. Dalen ML, Frøyland E, Saugstad OD, Mollnes TE, Rootwelt T. Post hypoxic hypothermia is protective in human NT2-N neurons regardless of oxygen concentration during reoxygenation. Brain Res 2009;1259:80–9.
  23. Markus T, Hansson S, Amer-Wåhlin I, Hellström-Westas L, Saugstad OD, Ley D. Cerebral inflammatory response after fetal asphyxia and hyperoxic resuscitation in newborn sheep. Pediatr Res 2007;62:71–7.
  24. Perez-Polo JR, Reilly CB, Rea HC. Oxygen resuscitation after hypoxia isch emia stimulates prostaglandin pathway in rat cortex. Int J Dev Neurosci 2011;29:639–44.
  25. Gill MB, Bockhorst K, Narayana P, Perez-Polo JR. Bax shuttling after neo natal hypoxia-ischemia: hyperoxia effects. J Neurosci Res 2008;86:3584
  27. Wollen EJ, Kwinta P, Bik-Multanowski M, et al. Hypoxia-reoxygenation affects whole-genome expression in the newborn eye. Invest Ophthalmol Vis Sci 2014;55:1393–401.
  28. Mojsilovic-Petrovic J, Callaghan D, Cui H, Dean C, Stanimirovic DB, Zhang W. Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes. J Neuroinflammation 2007;4:12.
  29. Ryter SW, Choi AM. Heme oxygenase-1: molecular mechanisms of gene expression in oxygen-related stress. Antioxid Redox Signal 2002;4:625–32.
  30. Bergeron M, Ferriero DM, Vreman HJ, Stevenson DK, Sharp FR. Hypoxia ischemia, but not hypoxia alone, induces the expression of heme oxy genase-1 (HSP32) in newborn rat brain. J Cereb Blood Flow Metab 1997;17:647–58.
  31. Buckley S, Warburton D. Dynamics of metalloproteinase-2 and -9, TGF beta, and uPA activities during normoxic vs. hyperoxic alveolarization. Am J Physiol Lung Cell Mol Physiol 2002;283:L747–54.
  32. Døhlen G, Antal EA, Castellheim A, Thaulow E, Kielland A, Saugstad OD. Hyperoxic resuscitation after hypoxia-ischemia induces cerebral inflam mation that is attenuated by tempol in a reporter mouse model with very young mice. J Perinat Med 2013;41:251–7.
  33. Dalen ML, Alme TN, Bjørås M, Munkeby BH, Rootwelt T, Saugstad OD. Reduced expression of DNA glycosylases in post-hypoxic newborn pigs undergoing therapeutic hypothermia. Brain Res 2010;1363:198–205.
  34. Bernaudin M, Tang Y, Reilly M, Petit E, Sharp FR. Brain genomic response following hypoxia and re-oxygenation in the neonatal rat. Identification of genes that might contribute to hypoxia-induced ischemic tolerance. J Biol Chem 2002;277:39728–38.
  35. Forsythe JA, Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996;16:4604–13.
  36. Vannucci SJ, Reinhart R, Maher F, et al. Alterations in GLUT1 and GLUT3 glucose transporter gene expression following unilateral hypoxia-ischemia in the immature rat brain. Brain Res Dev Brain Res 1998;107:255–64.
  37. Hagberg H, Gressens P, Mallard C. Inflammation during fetal and neonatal life: implications for neurologic and neuropsychiatric disease in children and adults. Ann Neurol 2012;71:444–57.
  38. Dommergues MA, Patkai J, Renauld JC, Evrard P, Gressens P. Proinflam matory cytokines and interleukin-9 exacerbate excitotoxic lesions of the newborn murine neopallium. Ann Neurol 2000;47:54–63.
  39. Morken TS, Widerøe M, Vogt C, et al. Longitudinal diffusion tensor and manganese-enhanced MRI detect delayed cerebral gray and white matter injury after hypoxia-ischemia and hyperoxia. Pediatr Res 2013;73:171–9.
  40. Shimabuku R, Ota A, Pereyra S, et al. Hyperoxia with 100% oxygen follow ing hypoxia-ischemia increases brain damage in newborn rats. Biol Neo nate 2005;88:168–71.
  41. Odd DE, Lewis G, Whitelaw A, Gunnell D. Resuscitation at birth and cog nition at 8 years of age: a cohort study. Lancet 2009;373:1615–22.
  42. Heidecker B, Kasper EK, Wittstein IS, et al. Transcriptomic biomark ers for individual risk assessment in new-onset heart failure. Circulation 2008;118:238–46.
  43. Lorek A, Takei Y, Cady EB, et al. Delayed (“secondary”) cerebral energy failure after acute hypoxia-ischemia in the newborn piglet: continuous 48-hour studies by phosphorus magnetic resonance spectroscopy. Pediatr Res 1994;36:699–706. Pediatric ReSeARCH 333 Copyright © 2015 International Pediatric Research Foundation, Inc. Volume 77 | Number 2 | February 2015