Development of the corpus callosum and cognition after neonatal encephalopathy.

OBJECTIVE: Neonatal imaging studies report corpus callosum abnormalities after neonatal hypoxic-ischaemic encephalopathy (HIE), but corpus callosum development and relation to cognition in childhood are unknown. Using magnetic resonance imaging (MRI), we examined the relationship between corpus callosum size, microstructure and cognitive and motor outcomes at early school-age children cooled for HIE (cases) without cerebral palsy compared to healthy, matched controls. A secondary aim was to examine the impact of HIE-related neonatal brain injury on corpus callosum size, microstructure and growth. METHODS: Participants aged 6-8 years underwent MRI, the Movement Assessment Battery for Children Second Edition and Wechsler Intelligence Scale for Children Fourth Edition. Cross-sectional area, volume, fractional anisotropy and radial diffusivity of the corpus callosum and five subdivisions were measured. Multivariable regression was used to assess associations between total motor score, full-scale IQ (FSIQ) and imaging metrics. RESULTS: Adjusting for age, sex and intracranial volume, cases (N = 40) compared to controls (N = 39) demonstrated reduced whole corpus callosum area (ß = -26.9, 95% confidence interval [CI] = -53.17, -0.58), volume (ß = -138.5, 95% CI = -267.54, -9.56), fractional anisotropy and increased radial diffusivity (P < 0.05) within segments II-V. In cases, segment V area (ß = 0.18, 95% CI = 0.004, 0.35), volume (ß = 0.04, 95% CI = 0.001, 0.079), whole corpus callosum fractional anisotropy (ß = 13.8 95% CI = 0.6, 27.1) and radial diffusivity (ß = -11.3, 95% CI = -22.22, -0.42) were associated with FSIQ. Growth of the corpus callosum was restricted in cases with a FSIQ ≤85, and volume was reduced in cases with mild neonatal multifocal injury compared to white matter injury alone. INTERPRETATION: Following neonatal HIE, morphological and microstructural changes in the corpus callosum are associated with reduced cognitive function at early school age.

Regulation of glutamate transport and neuroinflammation in a term newborn rat model of hypoxic-ischaemic brain injury.

In the newborn brain, moderate-severe hypoxia-ischaemia induces glutamate excitotoxicity and inflammation, possibly via dysregulation of candidate astrocytic glutamate transporter (Glt1) and pro-inflammatory cytokines (e.g. Tnfα, Il1ß, Il6). Epigenetic mechanisms may mediate dysregulation. Hypotheses: (1) hypoxia-ischaemia dysregulates mRNA expression of these candidate genes; (2) expression changes in Glt1 are mediated by DNA methylation changes; and (3) methylation values in brain and blood are correlated. Seven-day-old rat pups (n = 42) were assigned to nine groups based on treatment (for each timepoint: naïve (n = 3), sham (n = 3), hypoxia-ischaemia (n = 8) and timepoint for tissue collection (6, 12 and 24 h post-hypoxia). Moderate hypoxic-ischemic brain injury was induced via ligation of the left common carotid artery followed by 100 min hypoxia (8% O2, 36°C). mRNA was quantified in cortex and hippocampus for the candidate genes, myelin (Mbp), astrocytic (Gfap) and neuronal (Map2) markers (qPCR). DNA methylation was measured for Glt1 in cortex and blood (bisulphite pyrosequencing). Hypoxia-ischaemia induced pro-inflammatory cytokine upregulation in both brain regions at 6 h. This was accompanied by gene expression changes potentially indicating onset of astrogliosis and myelin injury. There were no significant changes in expression or promoter DNA methylation of Glt1. This pilot study supports accumulating evidence that hypoxia-ischaemia causes neuroinflammation in the newborn brain and prioritises further expression and DNA methylation analyses focusing on this pathway. Epigenetic blood biomarkers may facilitate identification of high-risk newborns at birth, maximising chances of neuroprotective interventions.

Glutamate Transport and Preterm Brain Injury.

Preterm birth complications are the leading cause of child death worldwide and a top global health priority. Among the survivors, the risk of life-long disabilities is high, including cerebral palsy and impairment of movement, cognition, and behavior. Understanding the molecular mechanisms of preterm brain injuries is at the core of future healthcare improvements. Glutamate excitotoxicity is a key mechanism in preterm brain injury, whereby the accumulation of extracellular glutamate damages the delicate immature oligodendrocytes and neurons, leading to the typical patterns of injury seen in the periventricular white matter. Glutamate excitotoxicity is thought to be induced by an interaction between environmental triggers of injury in the perinatal period, particularly cerebral hypoxia-ischemia and infection/inflammation, and developmental and genetic vulnerabilities. To avoid extracellular build-up of glutamate, the brain relies on rapid uptake by sodium-dependent glutamate transporters. Astrocytic excitatory amino acid transporter 2 (EAAT2) is responsible for up to 95% of glutamate clearance, and several lines of evidence suggest that it is essential for brain functioning. While in the adult EAAT2 is predominantly expressed by astrocytes, EAAT2 is transiently upregulated in the immature oligodendrocytes and selected neuronal populations during mid-late gestation, at the peak time for preterm brain injury. This developmental upregulation may interact with perinatal hypoxia-ischemia and infection/inflammation and contribute to the selective vulnerability of the immature oligodendrocytes and neurons in the preterm brain. Disruption of EAAT2 may involve not only altered expression but also impaired function with reversal of transport direction. Importantly, elevated EAAT2 levels have been found in the reactive astrocytes and macrophages of human infant post-mortem brains with severe white matter injury (cystic periventricular leukomalacia), potentially suggesting an adaptive mechanism against excitotoxicity. Interestingly, EAAT2 is suppressed in animal models of acute hypoxic-ischemic brain injury at term, pointing to an important and complex role in newborn brain injuries. Enhancement of EAAT2 expression and transport function is gathering attention as a potential therapeutic approach for a variety of adult disorders and awaits exploration in the context of the preterm brain injuries.

Feasibility of a Miniature Esophageal Heat Exchange Device for Rapid Therapeutic Cooling in Newborns: Preliminary Investigations in a Piglet Model.

Therapeutic hypothermia (TH) after neonatal encephalopathy, commonly provided by 72 hours of whole-body cooling using a wrap, limits parents' physical contact with their infants affecting bonding and may not be suitable for encephalopathic preterm infants with fragile skin. Alternative cooling methods are unavailable for this population. We investigated in a neonatal pig model the feasibility of achieving a 3.5°C reduction in rectal temperature (Trectal) similar to clinical TH protocols from 38.5°C (normothermia for pigs) to a target of 35°C ± 0.2°C, using a novel neonatal esophageal heat exchanger (NEHE), compared its efficacy to passive cooling, and investigated its ability to maintain target Trectal. Ventilated and anesthetized Landrace/Large white newborn pigs had the NEHE inserted. Water at adjustable temperatures and rates flowed down a central tube, returning up a surrounding distensible blind ending latex tube in a continuous loop. An initial experiment guided four subsequent cycles of passive cooling (30 minutes), rewarming to 38.5°C, active esophageal cooling to 35°C ± 0.2°C, active maintenance of target Trectal (30 minutes), and rewarming. We compared surface, rectal temperature, and hemodynamic changes among passive, active, and maintenance phases, and esophageal histopathology against control. Compared with passive cooling, esophageal cooling achieved target Trectal significantly earlier (71.3 minutes vs. 17.25 minutes, p = 0.003) with significantly greater rates of reduction in rectal (p = 0.0002) and surface (p = 0.005) temperatures and heart rate (p = 0.04). A water temperature of 39.1°C-40.2°C at a flow of 108-120 mL/min maintained Trectal around 35°C ± 0.2°C. The higher peak heart rate and blood pressure within 8 minutes of the maintenance phase (p = 0.04) subsequently stabilized. Histopathology showed congestion, edema, and neutrophil infiltration with increasing cycles. Esophageal cooling is feasible and effective in achieving rapid cooling in newborns. Subsequent maintenance at this temperature required continued circulation of warm water. Esophageal histopathology needs further evaluation after 72 hours servo-control cooling with a narrower range of water temperatures in a larger group of animals.

Survey of nutritional practices during therapeutic hypothermia for hypoxic-ischaemic encephalopathy.

OBJECTIVE: To evaluate current nutritional practices during and after therapeutic hypothermia (TH) for infants with hypoxic-ischaemic encephalopathy (HIE) in UK neonatal units. STUDY DESIGN: Email survey of neonatal clinicians. SETTING: UK neonatal units providing active TH. PATIENTS: Neonates cooled for HIE. METHODS: Email survey including questions regarding the timing of starting enteral feeds, volumes, frequency and parenteral nutrition (PN) use and availability of guidelines. RESULTS: Forty-nine responses were received (49/69, 71%). The rate of enteral feeding during TH and rewarming was 59% (29/49). There was a significant linear trend for the increase in the proportion of units starting enteral feeds (p=0.001) during TH. As compared with post-TH period, significantly lower milk volumes were started during TH (median (range): 7.5 mL/kg/day (1.5-24) vs 17.5 mL/kg/day (7.5-30), p=0.0004). During TH, breast milk was primarily used by 52% of units predominantly as 2-3 hourly feeds, and volumes were increased as tolerated in 55% of units. Only 29% (14/49) of units used PN, with 86% (12/14) of those offering enteral feeds during PN. Guidelines for feeding during TH were available in 31% (15/49) of units. CONCLUSIONS: Many neonatal clinicians offer enteral feeds predominantly using expressed breast milk, with or without PN, during TH, although with huge variability. The heterogeneity in the nutritional practice underscores the need for assessing the safety of both enteral and parenteral feeding during TH.

Low plasma magnesium is associated with impaired brain metabolism in neonates with hypoxic-ischaemic encephalopathy.

AIM: To determine the association between lowest plasma magnesium concentration and brain metabolism, and whether magnetic resonance imaging brain injury patterns moderated the association in hypoxic-ischemic encephalopathy. METHODS: In 131 early (day-of-life 3) and 65 late (day-of-life 10) scans of term encephalopathic infants born between 2004 and 2012, we examined the association of lowest plasma magnesium (until day-of-life 3) on basal ganglia and white matter peak metabolite ratios on magnetic resonance spectroscopy independent of covariates, stratified by the predominant patterns of injury (normal, basal nuclei/total, watershed, multifocal) using multiple linear regression. RESULTS: Lowest plasma magnesium was associated with lower white matter N-acetyl-aspartate/choline in the multifocal pattern on early scan (regression-coefficient, ß: 0.13; 95% CI: 0.04, 0.22) and in the basal nuclei/total pattern on late scan (ß: 0.08; 95% CI: 0.02, 0.15), and was negatively associated with basal ganglia lactate/N-acetyl-aspartate (ß: -0.16; 95% CI: -0.05, -0.28) and lactate/choline (ß: -0.1; 95% CI: -0.03, -0.17) ratio in the basal nuclei/total pattern on late scan independent of hypomagnesaemia correction, cooling and postmenstrual age at scan. Lowest plasma magnesium was not associated with metabolite ratios in other brain injury patterns. CONCLUSION: In infants with hypoxic-ischaemic encephalopathy, predominant patterns of brain injury moderated the association between lowest plasma magnesium in the first three days of life and impaired brain metabolism.

Association of Prenatal Diagnosis of Critical Congenital Heart Disease With Postnatal Brain Development and the Risk of Brain Injury.

IMPORTANCE: The relationship of prenatal diagnosis of critical congenital heart disease (CHD) with brain injury and brain development is unknown. Given limited improvement of CHD outcomes with prenatal diagnosis, the effect of prenatal diagnosis on brain health may reveal additional benefits. OBJECTIVE: To compare the prevalence of preoperative and postoperative brain injury and the trajectory of brain development in neonates with prenatal vs postnatal diagnosis of CHD. DESIGN, SETTING, AND PARTICIPANTS: Cohort study of term newborns with critical CHD recruited consecutively from 2001 to 2013 at the University of California, San Francisco and the University of British Columbia. Term newborns with critical CHD were studied with brain magnetic resonance imaging preoperatively and postoperatively to determine brain injury severity and microstructural brain development with diffusion tensor imaging by measuring fractional anisotropy and the apparent diffusion coefficient. Comparisons of magnetic resonance imaging findings and clinical variables were made between prenatal and postnatal diagnosis of critical CHD. A total of 153 patients with transposition of the great arteries and single ventricle physiology were included in this analysis. MAIN OUTCOMES AND MEASURES: The presence of brain injury on the preoperative brain magnetic resonance imaging and the trajectory of postnatal brain microstructural development. RESULTS: Among 153 patients (67% male), 96 had transposition of the great arteries and 57 had single ventricle physiology. The presence of brain injury was significantly higher in patients with postnatal diagnosis of critical CHD (41 of 86 [48%]) than in those with prenatal diagnosis (16 of 67 [24%]) (P = .003). Patients with prenatal diagnosis demonstrated faster brain development in white matter fractional anisotropy (rate of increase, 2.2%; 95% CI, 0.1%-4.2%; P = .04) and gray matter apparent diffusion coefficient (rate of decrease, 0.6%; 95% CI, 0.1%-1.2%; P = .02). Patients with prenatal diagnosis had lower birth weight (mean, 3184.5 g; 95% CI, 3050.3-3318.6) than those with postnatal diagnosis (mean, 3397.6 g; 95% CI, 3277.6-3517.6) (P = .02). Those with prenatal diagnosis had an earlier estimated gestational age at delivery (mean, 38.6 weeks; 95% CI, 38.2-38.9) than those with postnatal diagnosis (mean, 39.1 weeks; 95% CI, 38.8-39.5) (P = .03). CONCLUSIONS AND RELEVANCE: Newborns with prenatal diagnosis of single ventricle physiology and transposition of the great arteries demonstrate less preoperative brain injury and more robust microstructural brain development than those with postnatal diagnosis. These results are likely secondary to improved cardiovascular stability. The impact of these findings on neurodevelopmental outcomes warrants further study.

Reliability of Early Magnetic Resonance Imaging (MRI) and Necessity of Repeating MRI in Noncooled and Cooled Infants With Neonatal Encephalopathy.

In cooled newborns with encephalopathy, although late magnetic resonance imaging (MRI) scan (10-14 days of age) is reliable in predicting long-term outcome, it is unknown whether early scan (3-6 days of life) is. We compared the predominant pattern and extent of lesion between early and late MRI in 89 term neonates with neonatal encephalopathy. Forty-three neonates (48%) were cooled. The predominant pattern of lesions and the extent of lesion in the watershed region agreed near perfectly in noncooled (kappa = 0.94; k = 0.88) and cooled (k = 0.89; k = 0.87) infants respectively. There was perfect agreement in the extent of lesion in the basal nuclei in noncooled infants (k = 0.83) and excellent agreement in cooled infants (k = 0.67). Changes in extent of lesions on late MRI occurred in 19 of 89 infants, with higher risk in infants with hypoglycemia and moderate-severe lesions in basal nuclei. In most term neonates with neonatal encephalopathy, early MRI (relative to late scan) robustly predicts the predominant pattern and extent of injury.

Differential Tiam1/Rac1 activation in hippocampal and cortical neurons mediates differential spine shrinkage in response to oxygen/glucose deprivation.

Distinct neuronal populations show differential sensitivity to global ischemia, with hippocampal CA1 neurons showing greater vulnerability compared to cortical neurons. The mechanisms that underlie differential vulnerability are unclear, and we hypothesize that intrinsic differences in neuronal cell biology are involved. Dendritic spine morphology changes in response to ischemic insults in vivo, but cell type-specific differences and the molecular mechanisms leading to such morphologic changes are unexplored. To directly compare changes in spine size in response to oxygen/glucose deprivation (OGD) in cortical and hippocampal neurons, we used separate and equivalent cultures of each cell type. We show that cortical neurons exhibit significantly greater spine shrinkage compared to hippocampal neurons. Rac1 is a Rho-family GTPase that regulates the actin cytoskeleton and is involved in spine dynamics. We show that Rac1 and the Rac guanine nucleotide exchange factor (GEF) Tiam1 are differentially activated by OGD in hippocampal and cortical neurons. Hippocampal neurons express more Tiam1 than cortical neurons, and reducing Tiam1 expression in hippocampal neurons by shRNA enhances OGD-induced spine shrinkage. Tiam1 knockdown also reduces hippocampal neuronal vulnerability to OGD. This work defines fundamental differences in signalling pathways that regulate spine morphology in distinct neuronal populations that may have a role in the differential vulnerability to ischemia.

Therapeutic hypothermia delays the C-reactive protein response and suppresses white blood cell and platelet count in infants with neonatal encephalopathy.

BACKGROUND: Therapeutic hypothermia (HT) delays the cytokine response in infants with neonatal encephalopathy (NE). OBJECTIVE: To determine if HT delayed the C-reactive protein (CRP) response and altered white blood cell (WBC), neutrophil and platelet count course during the first week of life in infants with NE. DESIGN: Retrospective cohort study. SETTING: Regional neonatal intensive care unit, UK. PATIENTS: 104 term infants with NE (38 normothermia (NT) and 66 HT) born between 1998 and 2010. Infants not exposed to prenatal sepsis risk factors were classified as group 'A' and exposed infants to group 'B'. CRP >10 mg/L was defined as significant response. MAIN OUTCOME MEASURES: Time to CRP >10 mg/L, peak CRP, WBC, neutrophil and platelet count. RESULTS: Blood cultures were negative in all the infants. In babies who had CRP response, HT delayed time to CRP >10 mg/L (median (95% CI): group A, HT: 36 h (28.3 to 48.0); NT: 24 h (0.0 to 24.0); p=0.001; group B, HT: 30 h (15.2 to 56.8); NT: 12 h (0.0 to 24.0); p=0.009) and time to peak CRP (median (95% CI): group A, HT: 60 h (60.0 to 72.0); NT: 36 h (0.0 to 48.0); p=0.001; group B, HT: 84 h (62.1 to 120.0); NT: 24 h (0.0 to 36.0); p=0.001). Compared with NT, HT was associated with reduction in slope of CRP elevation by 0.5 (95% CI 0.04 to 0.97), WBC by 2.18×10(9)/L (95% CI 0.002 to 4.35) and platelet count by 32.3×10(9)/L (95% CI 2.75 to 61.8) independent of exposure to sepsis risk, meconium aspiration and severity of asphyxia. CONCLUSIONS: Therapeutic hypothermia delayed the initiation of CRP and its peak response, and depressed the WBC and platelet count compared with NT.

Effects of xenon and hypothermia on cerebrovascular pressure reactivity in newborn global hypoxic-ischemic pig model.

Autoregulation of cerebral perfusion is impaired in hypoxic-ischemic encephalopathy. We investigated whether cerebrovascular pressure reactivity (PRx), an element of cerebral autoregulation that is calculated as a moving correlation coefficient between averages of intracranial and mean arterial blood pressure (MABP) with values between -1 and +1, is impaired during and after a hypoxic-ischemic insult (HI) in newborn pigs. Associations between end-tidal CO2, seizures, neuropathology, and PRx were investigated. The effect of hypothermia (HT) and Xenon (Xe) on PRx was studied. Pigs were randomized to Sham, and after HI to normothermia (NT), HT, Xe or xenon hypothermia (XeHT). We defined PRx >0.2 as peak and negative PRx as preserved. Neuropathology scores after 72 hours of survival was grouped as 'severe' or 'mild.' Secondary PRx peak during recovery, predictive of severe neuropathology and associated with insult severity (P=0.05), was delayed in HT (11.5 hours) than in NT (6.5 hours) groups. Seizures were associated with impaired PRx in NT pigs (P=0.0002), but not in the HT/XeHT pigs. PRx was preserved during normocapnia and impaired during hypocapnia. Xenon abolished the secondary PRx peak, increased (mean (95% confidence interval (CI)) MABP (6.5 (3.8, 9.4) mm Hg) and cerebral perfusion pressure (5.9 (2.9, 8.9) mm Hg) and preserved the PRx (regression coefficient, -0.098 (95% CI (-0.18, -0.01)), independent of the insult severity.

Xenon offers stable haemodynamics independent of induced hypothermia after hypoxia-ischaemia in newborn pigs.

PURPOSE: To assess the effect of 18 hour (h) 50% xenon (Xe) inhalation at normothermia (NT, 38.5°C) or hypothermia (HT, 33.5°C) on mean arterial blood pressure (MABP), inotropic support and heart rate (HR) following an induced perinatal global hypoxic-ischaemic insult (HI) in newborn pigs. METHODS: Newborn pigs ventilated under inhalational anaesthesia, following a 45 min HI (inhaled oxygen fraction reduced until amplitude integrated electroencephalogram was less than 7 µV), were randomised to three Xe (n = 45) (50% Xe 18 h with NT, HT 12 h or HT 24 h) or three non-Xe groups (n = 53) (0% Xe with NT, HT 12 h or HT 24 h) under otherwise identical conditions. We measured MABP and HR every minute. Hypotension (MABP <40 mmHg) was treated sequentially with 2 × 10 mL/kg saline, dopamine, norepinephrine and hydrocortisone if required. RESULTS: Xe maintained higher MABP during HT (5.1 mmHg, 95% CI 2.34, 7.89), rewarming (10.1 mmHg, 95% CI 6.26, 13.95) and after cessation (4.1 mmHg, 95% CI 0.37, 7.84) independent of HT, inotropic support and acidosis. Xe reduced the duration of inotropic support by 12.6 h (95% CI 5.5, 19.73). Inotropic support decreased the HR reduction induced by HT from 9 to 5 bpm/°C during cooling and from 10-7 to 4-3 bpm/°C during rewarming. There was no interaction between Xe, HT, inotropic support and acidosis. Xe during HT cleared lactate faster; 3 h post-HI median (IQR) values of (Xe HT) 2.8 mmol/L (0.9, 3.1) vs. (HT) 5.9 mmol/L (2.5, 7.9), p = 0.0004. CONCLUSION: Xe maintained stable blood pressure, thereby reducing the inotropic support requirements during and after administration independently of induced HT-current neonatal encephalopathy treatment. Xe may offer haemodynamic benefits in clinical neuroprotection studies.

Multivariate analyses of factors that affect neonatal screening thyroid stimulating hormone.

AIMS: All screening programmes in the UK use a primary thyroid stimulating hormone (TSH) screen for congenital hypothyroidism. Recent attention has been paid to aspects of screening, such as the relation between blood spot TSH levels and birth weight or gestational age. The aim of our study was to determine the factors affecting screening neonatal TSH levels. METHODS: We conducted a retrospective analysis of blood spot screening TSH levels of all infants screened at a single regional screening laboratory. RESULTS: There were 6498 infants screened during a 12-week period. Screening TSH level showed negative correlation with gestational age and birth weight. Multiple linear regression analysis revealed low birth weight as the only independent factor affecting screening TSH level. CONCLUSIONS: Low birth weight infants appear to be at risk of thyroidal dysfunction. Our study showed that there were clinically significant but weak correlation between higher screening TSH levels and low birth weight. The clinical importance of these findings requires larger prospective studies to further elucidate the relevance of these factors affecting TSH screening levels.

Xenon enhances hypothermic neuroprotection in asphyxiated newborn pigs.

OBJECTIVE: To investigate whether inhaling 50% xenon during hypothermia (HT) offers better neuroprotection than xenon or HT alone. METHODS: Ninety-eight newborn pigs underwent a 45-minute global hypoxic-ischemic insult severe enough to cause permanent brain injury, and 12 pigs underwent sham protocol. Pigs then received intravenous anesthesia and were randomized to 6 treatment groups: (1) normothermia (NT; rectal temperature 38.5 degrees C, n = 18); (2) 18 hours 50% xenon with NT (n = 12); (3) 12 hours HT (rectal temperature 33.5 degrees C, n = 18); (4) 24 hours HT (rectal temperature 33.5 degrees C, n = 17); (5) 18 hours 50% xenon with 12 hours HT (n = 18); and (6) 18 hours 50% xenon with 24 hours HT (n = 17). Fifty percent xenon was administered via a closed circle with 30% oxygen and 20% nitrogen. After 10 hours rewarming, cooled pigs remained normothermic until terminal perfusion fixation at 72 hours. Global and regional brain neuropathology and clinical neurological scores were performed. RESULTS: Xenon (p = 0.011) and 12 or 24 hours HT (p = 0.003) treatments offered significant histological global, and regional neuroprotection. Combining xenon with HT yielded an additive neuroprotective effect, as there was no interaction effect (p = 0.54). Combining Xenon with 24 hours HT offered 75% global histological neuroprotection with similarly improved regional neuroprotection: thalamus (100%), brainstem (100%), white matter (86%), basal ganglia (76%), cortical gray matter (74%), cerebellum (73%), and hippocampus (72%). Neurology scores improved in the 24-hour HT and combined xenon HT groups at 72 hours. INTERPRETATION: Combining xenon with HT is a promising therapy for severely encephalopathic infants, doubling the neuroprotection offered by HT alone.