Gene expression in the intestine of newborn piglets after hypoxia-reoxygenation.

BACKGROUND: In preterm infants, intestinal hypoxia may partly contribute to the pathophysiology of necrotizing enterocolitis through changes in gene expression. Splanchnic hypoxia can be detected with monitoring of regional splanchnic oxygen saturation (rsSO2). Using a piglet model of asphyxia, we aimed to correlate changes in rsSO2 to gene expression. METHODS: Forty-two newborn piglets were randomized to control or intervention groups. Intervention groups were subjected to hypoxia until they were acidotic and hypotensive. Next, they were reoxygenated for 30 min according to randomization, i.e., 21% O2, 100% O2, or 100% O2 for 3 min followed by 21% O2, and observed for 9 h. We continuously measured rsSO2 and calculated mean rsSO2 and variability of rsSO2 (rsCoVar = SD/mean). Samples of terminal ileum were analyzed for mRNA expression of selected genes related to inflammation, erythropoiesis, fatty acid metabolism, and apoptosis. RESULTS: The expression of selected genes was not significantly different between control and intervention groups. No associations between mean rsSO2 and gene expression were observed. However, lower rsCoVar was associated with the upregulation of apoptotic genes and the downregulation of inflammatory genes (P < 0.05). CONCLUSION: Our study suggests that hypoxia and reoxygenation cause reduced vascular adaptability, which seems to be associated with the upregulation of apoptosis and downregulation of inflammation. IMPACT: Our results provide important insight into the (patho)physiological significance of changes in the variability of rsSO2. Our findings may advance future research and clinical practice regarding resuscitation strategies of preterm infants.

Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia.

BACKGROUND: Since the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRT-PCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA. METHODS AND RESULTS: Newborn piglets (n = 19) were either exposed to hypoxia (n = 11) or were part of the sham-operated control group (n = 8). Blood samples were collected at baseline (= start) and at the end of hypoxia or at 40-45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for ß-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group. CONCLUSIONS: Both, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses.

Splanchnic oxygen saturation during reoxygenation with 21% or 100% O2 in newborn piglets.

BACKGROUND: Increasing evidence recognizes the harm of excess oxygen to lungs, eyes, and brain of preterm infants, but not yet to the intestine. We assessed changes in splanchnic oxygenation during reoxygenation with 21% compared to 100% O2 in a newborn piglet model of perinatal asphyxia. METHODS: We randomized 25 piglets to control or intervention. Intervention groups underwent global hypoxia until acidosis and hypotension occurred. Piglets were reoxygenated for 30 min with 21% or 100% O2 and observed for 9 h. We continuously measured regional splanchnic oxygen saturation (rsSO2) using near-infrared spectroscopy (NIRS). We calculated mean rsSO2 and rsCoVar (as SD/mean). We measured PaO2 and SaO2, sampled from the right carotid artery. RESULTS: Reoxygenation after global hypoxia restored rsSO2. Reoxygenation with 100% O2 increased rsSO2 to values significantly higher than baseline. In intervention groups, rsCoVar decreased during observation compared to baseline. We found a correlation between rsSO2 and PaO2 (r = 0.420, P < 0.001) and between rsSO2 and SaO2 (r = 0.648, P < 0.001) in pooled data from the entire experiment. CONCLUSION: Reoxygenation after global hypoxia improves splanchnic oxygenation, but is associated with reduced variability of rsSO2. Reoxygenation with 100% O2 exposes the intestine to hyperoxia. Splanchnic NIRS is able to detect intestinal hypoxia and hyperoxia. IMPACT: Splanchnic oxygenation improves during reoxygenation after global hypoxia, though reoxygenation with 100% O2 exposes the intestine to hyperoxia. Decreased variability of splanchnic oxygenation several hours after hypoxia and reoxygenation seems to be independent of the resuscitation strategy, and may indicate intestinal injury. Splanchnic NIRS monitoring was able to detect intestinal hypoxia and exposure to hyperoxia, as evidenced by a strong correlation between splanchnic oxygenation and arterial oxygen content.

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

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.

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

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.

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

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% O2 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.

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

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.

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

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.

Long Non-Coding RNAs in Hypoxia and Oxidative Stress: Novel Insights Investigating a Piglet Model of Perinatal Asphyxia.

Birth asphyxia is the leading cause of death and disability in young children worldwide. Long non-coding RNAs (lncRNAs) may provide novel targets and intervention strategies due to their regulatory potential, as demonstrated in various diseases and conditions. We investigated cardinal lncRNAs involved in oxidative stress, hypoxia, apoptosis, and DNA damage using a piglet model of perinatal asphyxia. A total of 42 newborn piglets were randomized into 4 study arms: (1) hypoxia-normoxic reoxygenation, (2) hypoxia-3 min of hyperoxic reoxygenation, (3) hypoxia-30 min of hyperoxic reoxygenation, and (4) sham-operated controls. The expression of lncRNAs BDNF-AS, H19, MALAT1, ANRIL, TUG1, and PANDA, together with the related target genes VEGFA, BDNF, TP53, HIF1α, and TNFα, was assessed in the cortex, the hippocampus, the white matter, and the cerebellum using qPCR and Droplet Digital PCR. Exposure to hypoxia-reoxygenation significantly altered the transcription levels of BDNF-AS, H19, MALAT1, and ANRIL. BDNF-AS levels were significantly enhanced after both hypoxia and subsequent hyperoxic reoxygenation, 8% and 100% O2, respectively. Our observations suggest an emerging role for lncRNAs as part of the molecular response to hypoxia-induced damages during perinatal asphyxia. A better understanding of the regulatory properties of BDNF-AS and other lncRNAs may reveal novel targets and intervention strategies in the future.

A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation.

Neonatal piglets have been extensively used as translational models for perinatal asphyxia. In 2007, we adapted a well-established piglet asphyxia model by introducing cardiac arrest. This enabled us to study the impact of severe asphyxia on key outcomes, including the time taken for the return of spontaneous circulation (ROSC), as well as the effect of chest compressions according to alternative protocols for cardiopulmonary resuscitation. Due to the anatomical and physiological similarities between piglets and human neonates, piglets serve as good models in studies of cardiopulmonary resuscitation and hemodynamic monitoring. In fact, this cardiac arrest model has provided evidence for guideline development through research on resuscitation protocols, pathophysiology, biomarkers, and novel methods for hemodynamic monitoring. Notably, the incidental finding that a substantial fraction of piglets have pulseless electrical activity (PEA) during cardiac arrest may increase the applicability of the model (i.e., it may be used to study pathophysiology extending beyond the perinatal period). However, the model generation is technically challenging and requires various skill sets, dedicated personnel, and a fine balance of the measures, including the surgical protocols and the use of sedatives/analgesics, to ensure a reasonable rate of survival. In this paper, the protocol is described in detail, as well as experiences with adaptations to the protocol over the years.

Associations between unit workloads and outcomes of first extubation attempts in extremely premature infants below a gestational age of 26 weeks.

Objective: The objective was to explore whether high workloads in neonatal intensive care units were associated with short-term respiratory outcomes of extremely premature (EP) infants born <26 weeks of gestational age. Methods: This was a population-based study using data from the Norwegian Neonatal Network supplemented by data extracted from the medical records of EP infants <26 weeks GA born from 2013 to 2018. To describe the unit workloads, measurements of daily patient volume and unit acuity at each NICU were used. The effect of weekend and summer holiday was also explored. Results: We analyzed 316 first planned extubation attempts. There were no associations between unit workloads and the duration of mechanical ventilation until each infant's first extubation or the outcomes of these attempts. Additionally, there were no weekend or summer holiday effects on the outcomes explored. Workloads did not affect the causes of reintubation for infants who failed their first extubation attempt. Conclusion: Our finding that there was no association between the organizational factors explored and short-term respiratory outcomes can be interpreted as indicating resilience in Norwegian neonatal intensive care units.

Neonatal Impedance Cardiography in Asphyxiated Piglets-A Feasibility Study.

Objectives: Impedance cardiography (ICG) is a non-invasive method for continuous cardiac output measurement and has the potential to improve monitoring and treatment of sick neonates. PhysioFlow® is a signal-morphology ICG-system showing promising results in adults with low and high cardiac output, but no data from neonates or neonatal models exist. The aim of this study was to investigate PhysioFlow® feasibility in asphyxiated newborn piglets. Methods: Fifteen piglets, under continuous arterial heart rate (HR) and blood pressure (BP) monitoring, were asphyxiated until asystole. Cardiopulmonary resuscitation was performed and the piglets monitored after return of spontaneous circulation (ROSC). Arterial lactate was measured at baseline, every 5 min throughout asphyxiation, at asystole, and at 10 min and later every 30 min after ROSC. PhysioFlow® measured cardiac stroke volume (SV) and HR, and calculated cardiac index (CI) (L/m2/min). Registrations with a signal quality < 75% were excluded, and registrations recorded for 30 min from start of asphyxia analyzed. Pearson correlations were calculated for CI; and HR, mean BP and blood lactate. Results: The piglets were asphyxiated for median (interquartile range) 30 (20-35) min and had a lactate at asystole of 15.0 (9.1-17.0) mmol/L. Out of a total of 20.991 registrations in all animals combined, there were 10.148 (48.3%) registrations with a signal quality ≥ 75%. Signal quality ≥ 75% varied in individual piglets from 7 to 82% of registrations. We analyzed 1.254 registrations recorded 30 min from initiation of asphyxia, i.e., in piglets with brief asphyxia times, this included cardiopulmonary resuscitation and post-ROSC observation. There was a positive correlation between CI and SVI (r = 0.90, p < 0.001), and between CI and HR (r = 0.446, p < 0.001). There was no correlation between CI, or mean BP or lactate (p = 0.98 and 0.51, respectively). Conclusion: About half of ICG-registrations in asphyxiated piglets were of good quality. However, signal quality was highly variable between piglets. In total, there was a higher proportion of reliable ICG-registrations than reported from clinical delivery room studies using electrical velocimetry. Our data are physiologically plausible and supports further research evaluating PhysioFlow® for cardiac output monitoring in perinatal asphyxia. In particular, factors influencing inter-individual variations in signal quality should be explored.

Predictors of extubation success: a population-based study of neonates below a gestational age of 26 weeks.

OBJECTIVE: The aim of the study was to investigate first extubation attempts among extremely premature (EP) infants and to explore factors that may increase the quality of clinical judgement of extubation readiness. DESIGN AND METHOD: A population-based study was conducted to explore first extubation attempts for EP infants born before a gestational age (GA) of 26 weeks in Norway between 1 January 2013 and 31 December 2018. Eligible infants were identified via the Norwegian Neonatal Network database. The primary outcome was successful extubation, defined as no reintubation within 72 hours after extubation. RESULTS: Among 482 eligible infants, 316 first extubation attempts were identified. Overall, 173 (55%) infants were successfully extubated, whereas the first attempt failed in 143 (45%) infants. A total of 261 (83%) infants were extubated from conventional ventilation (CV), and 55 (17%) infants were extubated from high-frequency oscillatory ventilation (HFOV). In extubation from CV, pre-extubation fraction of inspired oxygen (FiO2) ≤0.35, higher Apgar score, higher GA, female sex and higher postnatal age were important predictors of successful extubation. In extubation from HFOV, a pre-extubation FiO2 level ≤0.35 was a relevant predictor of successful extubation. CONCLUSIONS: The correct timing of extubation in EP infants is important. In this national cohort, 55% of the first extubation attempts were successful. Our results suggest that additional emphasis on oxygen requirement, sex and general condition at birth may further increase extubation success when clinicians are about to extubate EP infants for the first time.

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

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.

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

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.

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

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-1ß 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.

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

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 [18F]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 (FiO2)0.08 until base excess -20 mmol/L and/or a mean arterial blood pressure decrease to 20 mm Hg, followed by resuscitation with FiO2 0.21 or 1.0. Three piglets served as controls and one had intracranial injection of lipopolysaccharide (LPS). Whole body [18F]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 ±â€¯0.10 and 1.71 ±â€¯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 [18F]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 [18F]GE180. IMPLICATIONS FOR PATIENT CARE: TSPO-PET with [18F]GE180 might not be suitable for early detection of perinatal hypoxic ischemic brain injury.

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

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.

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

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-1ß 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.