Prognostic Neurobiomarkers in Neonatal Encephalopathy.

Therapeutic hypothermia (TH) is now a standard treatment for infants with moderate-to-severe neonatal encephalopathy (NE), and improves brain damage on neuroimaging and neurodevelopmental outcomes. Critically, for effective neuroprotection, hypothermia should be started within 6 h from birth. There is compelling evidence to suggest that a proportion of infants with mild NE have material risk of developing brain damage and poor outcomes. This cohort is increasingly being offered TH, despite lack of trial evidence for its benefit. In current practice, infants need to be diagnosed within 6 h of birth for therapeutic treatment, compared to retrospective NE grading in the pre-hypothermia era. This presents challenges as NE is a dynamic brain disorder that can worsen or resolve over time. Neurological symptoms of NE can be difficult to discern in the first few hours after birth, and confounded by analgesics and anesthetic treatment. Using current enrolment criteria, a significant number of infants with NE that would benefit from hypothermia are not treated, and vice versa, some infants receive hypothermia when its benefit will be limited. Better biomarkers are needed to further improve management and treatment of these neonates. In the present review, we examine the latest research, and highlight a central limitation of most current biomarkers: that their predictive value is consistently greatest after most neuroprotective therapies are no longer effective.

Tumor necrosis factor inhibition attenuates white matter gliosis after systemic inflammation in preterm fetal sheep.

BACKGROUND: Increased circulating levels of tumor necrosis factor (TNF) are associated with greater risk of impaired neurodevelopment after preterm birth. In this study, we tested the hypothesis that systemic TNF inhibition, using the soluble TNF receptor Etanercept, would attenuate neuroinflammation in preterm fetal sheep exposed to lipopolysaccharide (LPS). METHODS: Chronically instrumented preterm fetal sheep at 0.7 of gestation were randomly assigned to receive saline (control; n = 7), LPS infusion (100 ng/kg i.v. over 24 h then 250 ng/kg/24 h for 96 h plus 1 µg LPS boluses at 48, 72, and 96 h, to induce inflammation; n = 8) or LPS plus two i.v. infusions of Etanercept (2 doses, 5 mg/kg infused over 30 min, 48 h apart) started immediately before LPS-exposure (n = 8). Sheep were killed 10 days after starting infusions, for histology. RESULTS: LPS boluses were associated with increased circulating TNF, interleukin (IL)-6 and IL-10, electroencephalogram (EEG) suppression, hypotension, tachycardia, and increased carotid artery perfusion (P < 0.05 vs. control). In the periventricular and intragyral white matter, LPS exposure increased gliosis, TNF-positive cells, total oligodendrocytes, and cell proliferation (P < 0.05 vs control), but did not affect myelin expression or numbers of neurons in the cortex and subcortical regions. Etanercept delayed the rise in circulating IL-6, prolonged the increase in IL-10 (P < 0.05 vs. LPS), and attenuated EEG suppression, hypotension, and tachycardia after LPS boluses. Histologically, Etanercept normalized LPS-induced gliosis, and increase in TNF-positive cells, proliferation, and total oligodendrocytes. CONCLUSION: TNF inhibition markedly attenuated white matter gliosis but did not affect mature oligodendrocytes after prolonged systemic inflammation in preterm fetal sheep. Further studies of long-term brain maturation are now needed.

Ontogeny and control of the heart rate power spectrum in the last third of gestation in fetal sheep.

Power spectral analysis of fetal heart rate variability has been proposed to provide a non-invasive estimate of autonomic balance. However, there are few systematic data before birth. We therefore examined developmental changes in the frequency power spectrum at very low (0-0.04 Hz), low (0.04-0.15 Hz) and high frequencies (0.15-0.4 Hz), as well as the ratio of low- to high-frequency power (LF/HF), in chronically catheterized, healthy fetal sheep at 0.6 (n = 8), 0.7 (n = 7) and 0.8 gestational age (ga; n = 11). In a second study, 0.8 ga fetuses received either atropine (4.8 mg bolus, then 4.8 mg h(-1) for 30 min, n = 6) or 6-hydroxydopamine (20 mg ml(-1) at 2.5 ml h(-1) for 3 h; n = 9). Data were analysed by sleep state, defined by low-voltage-high-frequency (LV) or high-voltage-low-frequency (HV) EEG. Total spectral power increased with gestational age (P < 0.05), while LF/HF decreased from 0.6 to 0.7 ga. At 0.8 ga, heart rate and LF/HF were significantly higher during HV than LV sleep (P < 0.05). Consistent with this, although total spectral power was not significantly greater during HV sleep, there was a significant interaction between sleep state and frequency band (P = 0.02). Both atropine (P = 0.05) and 6-hydroxydopamine (P < 0.05) were associated with an overall reduction in spectral power but no significant effect on the LF/HF ratio. This study does not support substantial, consistent differences between the frequencies of sympathetic and parasympathetic activity in late-gestation fetal sheep.

What brakes the preterm brain? An arresting story.

Children surviving premature birth have a high risk of cognitive and learning disabilities and attention deficit. In turn, adverse outcomes are associated with persistent reductions in cerebral growth on magnetic resonance imaging (MRI). It is striking that modern care has been associated with a dramatic reduction in the risk of cystic white matter damage, but modest improvements in terms of neurodevelopmental impairment. This review will explore the hypothesis that the disability is primarily associated with impaired neural connectivity rather than cell death alone. Very preterm infants exhibit reduced thalamocortical connectivity and cortical neuroplasticity compared with term-born controls. In preterm fetal sheep, moderate cerebral ischemia with no neuronal loss, but significant diffuse failure of maturation of cortical pyramidal neurons, was associated with impaired dendritic growth and synapse formation, consistent with altered connectivity. These changes were associated with delayed decline in cortical fractional anisotropy (FA) on MRI. Supporting these preclinical findings, preterm human survivors showed similar enduring impairment of microstructural development of the cerebral cortex defined by FA, consistent with delayed formation of neuronal processes. These findings offer the promise that better understanding of impairment of neural connectivity may allow us to promote normal development and growth of the cortex after preterm birth.

Astrocytes and microglia in acute cerebral injury underlying cerebral palsy associated with preterm birth.

Cerebral palsy is one of the most devastating consequences of brain injury around the time of birth, and nearly a third of cases are now associated with premature birth. Compared with term babies, preterm babies have an increased incidence of complications that may increase the risk of disability, such as intraventricular hemorrhage, periventricular leukomalacia, sepsis, and necrotizing enterocolitis. The response to injury is highly dependent on brain maturity, and although cellular vulnerability is well documented, there is now evidence that premyelinating axons are also particularly sensitive to ischemic injury. In this review, we will explore recent evidence highlighting a central role for glia in mediating increased risk of disability in premature infants, including excessive activation of microglia and opening of astrocytic gap junction hemichannels in spreading injury after brain ischemia, in part likely involving release of adenosine triphosphate (ATP) and overactivation of purinergic receptors, particularly in white matter. We propose the hypothesis that inflammation-induced opening of connexin hemichannels is a key regulating event that initiates a vicious circle of excessive ATP release, which in turn propagates activation of purinergic receptors on microglia and astrocytes. This suggests that developing effective neuroprotective strategies for preterm infants requires a detailed understanding of glial responses.

Connexin hemichannel blockade improves outcomes in a model of fetal ischemia.

OBJECTIVE: Connexin hemichannels can open during ischemia, resulting in loss of membrane potential, calcium influx, and release of glutamate. In this study, we tested the hypothesis that opening of hemichannels after cerebral ischemia may contribute to delayed evolution of injury. METHODS: We infused a mimetic peptide that blocks connexin 43 hemichannels into the lateral ventricle of chronically instrumented fetal sheep in utero at 128 ± 1 days gestation (term is 147 days), starting 90 minutes after 30 minutes of severe ischemia induced by reversible bilateral carotid artery occlusion, for either 1 or 25 hours. Sheep were killed 7 days later. RESULTS: Peptide infusion was associated with a graded improvement in recovery of electroencephalographic power after 7 days recovery, from -13 ± 1.9 dB (n = 7) after ischemia-vehicle to -9 ± 1.6 dB (n = 7) after ischemia-short infusion and -5 ± 1.6 dB after ischemia-long infusion (n = 6, p < 0.05). Peptide infusion was associated with reduced seizure activity after ischemia, less frequent status epilepticus (p < 0.05), and earlier return of sleep state cycling (p < 0.05). Ischemia-long infusion (but not ischemia-short infusion) was associated with improved survival of oligodendrocytes in intragyral and periventricular white matter (p < 0.05) and increased brain weight (p < 0.05). Ischemia-long infusion was associated with an intermediate estimate of surviving neurons in the parasagittal cortex of 2.9 ± 0.8 × 10(6), in comparison to sham control (4.3 ± 0.9 × 10(6)) or ischemia-vehicle (1.5 ± 0.4 × 10(6); p < 0.05 vs sham control). INTERPRETATION: These data support the hypothesis that opening of connexin hemichannels is a significant mediator of postischemic white and gray matter dysfunction and injury.

Cell therapy for neonatal hypoxia-ischemia and cerebral palsy.

Perinatal hypoxic-ischemic brain injury remains a major cause of cerebral palsy. Although therapeutic hypothermia is now established to improve recovery from hypoxia-ischemia (HI) at term, many infants continue to survive with disability, and hypothermia has not yet been tested in preterm infants. There is increasing evidence from in vitro and in vivo preclinical studies that stem/progenitor cells may have multiple beneficial effects on outcome after hypoxic-ischemic injury. Stem/progenitor cells have shown great promise in animal studies in decreasing neurological impairment; however, the mechanisms of action of stem cells, and the optimal type, dose, and method of administration remain surprisingly unclear, and some studies have found no benefit. Although cell-based interventions after completion of the majority of secondary cell death appear to have potential to improve functional outcome for neonates after HI, further rigorous testing in translational animal models is required before randomized controlled trials should be considered.

Dopamine infusion for postresuscitation blood pressure support after profound asphyxia in near-term fetal sheep.

Dopamine is commonly used for blood pressure support in the neonate, but has limited empirical evidence to support its use. We tested the hypothesis that after near-terminal asphyxia in utero, dopamine infusions would prevent secondary hypotension. Fetal sheep (122-129 days of gestation; term is 147 days) received umbilical cord occlusion for 15 min or sham occlusion (n = 5). If the mean arterial blood pressure fell below 90% of baseline within 6 h after occlusion, fetuses were randomized to either dopamine infusion starting at 4 µg kg(-1) min(-1) and titrated according to mean arterial blood pressure up to a maximum of 40 µg kg(-1) min(-1) (n = 5) or to the same volume of normal saline (n = 5). Dopamine infusion, initiated at a median of 180 min after occlusion (range 96-280 min), was associated with a marked but transient increase in mean arterial blood pressure and fall in femoral blood flow compared with saline. Terminal hypotension developed later in four of the five fetuses that received maximal dopamine infusions than in five of five receiving saline infusion [517 (range 240-715) versus 106 min (range 23-497) after the start of infusions, P < 0.05]. In conclusion, dopamine infusion delayed but did not prevent terminal hypotension after severe asphyxia.

Neural plasticity and the Kennard principle: Does it work for the preterm brain?

The Kennard principle suggests that the immature brain should be more able to recover from injury than the more developed brain. Curiously, preterm infants continue to have a high rate of debilitating neurodevelopmental handicaps despite a progressive improvement in structural damage to the brain, from acute necrotic injury of the periventricular white matter, with axonal loss in historical cohorts, to diffuse gliosis with trivial axonal damage. In the present review we examine recent evidence that disability after preterm birth is largely mediated by disturbed development of neuronal connections. Potential mechanisms include impaired white matter maturation associated with gliosis, suboptimal neuronal maturation, adverse effects of infection/inflammation on the cell environment, exposure to clinical therapies that modulate brain function (including maternal glucocorticoids), upregulation of physiological apoptosis and loss or misprogramming of progenitor cells in the subventricular zone. These findings suggest that insults during this critical phase alter the trajectory of brain development and that a key focus of basic science and clinical research should be to understand neuronal connectivity, as well as the triggers of cell death.

2D Wavelet-Scalogram Deep-Learning for Seizures Pattern Identification in the Post-Hypoxic-Ischemic EEG of Preterm Fetal Sheep.

Neonatal seizures after an hypoxic-ischemic (HI) event in preterm newborns can contribute to neural injury and cause impaired brain development. Preterm neonatal seizures are often not detected or their occurrence underestimated. Therefore, there is a need to improve knowledge about preterm seizures that can help establish diagnostic tools for accurate identification of seizures and for determining morphological differences. We have previously shown the superior utility of deep-learning algorithms for the accurate identification and quantification of post-HI microscale epileptiform transients (e.g., gamma spikes and sharp waves) in preterm fetal sheep models; before the irreversible secondary phase of cerebral energy failure starts by the bursts of high-amplitude stereotypic evolving seizures (HAS) in the signal. We have previously developed successful deep-learning algorithms that accurately identify and quantify the micro-scale transients, during the latent phase. Building up on our deep-learning strategies, this work introduces a real-time deep-learning-based pattern fusion approach to identify HAS in the 256Hz sampled post-HI data from our preterm fetuses. Here, for the first time, we propose a 17-layer deep convolutional neural network (CNN) classifier fed with 2D wavelet-scalogram (WS) images of the EEG patterns for accurate seizure identification. The WS-CNN classifier was cross-validated over 1812 manually annotated EEG segments during ~6 to 48 hours post-HI recordings. The classifier accurately recognized HAS patterns with 97.19% overall accuracy (AUC = 0.96).Clinical relevance-The promising results from this preliminary work indicate the ability of the proposed WS-CNN pattern classifier to identify HI-related seizures in the neonatal preterm brain using 256Hz EEG; the frequency commonly used clinically for data collection.

Seven- to eight-year follow-up of the CoolCap trial of head cooling for neonatal encephalopathy.

INTRODUCTION: We sought to determine whether 18- to 22-mo neurodevelopmental outcomes predicted functional outcomes at 7-8 y for survivors of the CoolCap study of therapeutic hypothermia for neonates with hypoxic-ischemic encephalopathy. RESULTS: WeeFIM ratings were completed at 7-8 y of age on 62 (32 cooled; 30 standard care) of 135 surviving children who had had neurodevelopmental assessment at 18 mo. There was 1 refusal, 58 lost to follow-up, and 14 children whose centers declined to participate. Disability status at 18 mo was strongly associated with WeeFIM ratings (P < 0.001); there was no significant effect of treatment (P = 0.83). DISCUSSION: Functional outcome at 7-8 y of survivors of neonatal encephalopathy is associated with 18-mo neurodevelopmental assessment, supporting the long-term predictive value of a favorable outcome at 18 mo assessed by published trials of therapeutic hypothermia. METHODS: All surviving children who participated in the CoolCap study and were assessed at 18 mo were eligible for reassessment using the WeeFIM instrument that qualitatively measures self-care, mobility, and cognitive function. Center investigators obtained consent from the families for a certified researcher to administer the WeeFIM instrument by phone.

The incidence, clinical features, and treatment of type 2 diabetes in children <15 yr in a population-based cohort from Auckland, New Zealand, 1995­2007.

BACKGROUND: The incidence of type 2 diabetes mellitus (T2DM) is increasing in adolescents in most western countries. The time-course of glycemic control and impact of early treatment remain poorly understood. OBJECTIVES: To determine the change in incidence of T2DM, and the time-course of glycemic control in a regional pediatric cohort with T2DM. METHODS: Retrospective analysis of prospectively collected data on 52 patients with T2DM from a population-based treatment referral cohort from 1 January 1995 to 31 December 2007. RESULTS: The annual incidence of new cases of T2DM in children <15 yr increased fivefold in the Auckland region of New Zealand from 1995 [0.5/100,000; 95% confidence interval (CI) 0.0­2.2] to 2007 (2.5/100,000; 95% CI 1.0­5.5). The average annual incidence per 100,000 over the entire period was 1.3 (95% CI 1.0­1.8) overall, 0.1 (0.0­0.4) in Europeans, and 3.4 in both Maori (2.0­5.3) and Pacifica (2.2­5.0). Fifty-seven percent of children were symptomatic at presentation. Fifty-eight percent of patients were treated with insulin from diagnosis, most of whom were symptomatic (p = 0.003). Follow-up data were available for 48 patients with a mean of 2.4 yr. Although insulin therapy was associated with a greater fall in HbA1c values in the first 12 months of treatment (to a nadir of 7.1 vs. 8.1%, p < 0.05), there was a rapid deterioration after 12 months, and subsequent mean HbA1c values were >9% in both groups. Therapy did not affect body mass index standard deviation score (BMI SDS). CONCLUSIONS: The incidence of T2DM in childhood or adolescence increased markedly over a 13-yr period in the Auckland region. Long-

Missed congenital hypothyroidism in an identical twin.

Newborn screening for congenital hypothyroidism has been remarkably effective, although rare cases of false negative screening have been reported in same sex twins, presumptively due to fetal blood exchange. We report a case in which the diagnosis of congenital hypothyroidism due to thyroid ectopia in a monozygotic twin was delayed by 8 months, with a normal newborn screening TSH level of 11 mIU/L blood (normal < 15 mIU/L) at 2 days of life. This is the first such case since the national New Zealand newborn screening programme introduced screening for congenital hypothyroidism in 1981 (30 years ago). Repeating thyroid studies at 14 days of age in same-sex twins has been advocated to avoid delayed diagnosis, but given the low risk, may not be cost effective. It is important to maintain a high index of suspicion in same-sex twin pregnancies of potential congenital hypothyroidism.

Impact of insulin pumps on glycaemic control in a pump-naïve paediatric regional population.

AIM: To examine the clinical impact of insulin-pump therapy for children with type 1 diabetes mellitus (T1DM) in a regional paediatric service, Auckland, New Zealand. METHODS: Retrospective analysis of children with T1DM from the Starship paediatric diabetes database who started on insulin-pump therapy from 2002 to 2008 compared with the whole T1DM population and with an equal number of non-pump patients matched by age, sex, ethnicity and duration of diabetes. RESULTS: From 621 subjects with 6680 clinic visits, 75 children were treated with insulin-pump therapy for more than 12 months. Transitioning to insulin-pump treatment was associated with an improvement in HbA1c compared with baseline (-0.3%/year, P < 0.001) for up to 3 years. In contrast, despite similar deprivation scores, non-pump controls showed a continuing trend to higher HbA1C values (+0.2%/year, P < 0.01). The risk of severe hypoglycaemia fell after pump start (from 27 (0-223) to 5 (0-0.91) events/100 patient years) with no change in non-pump controls; the rate of diabetic ketoacidosis remained low in both groups. CONCLUSIONS: In a pump-naïve regional paediatric population, insulin-pump therapy for T1DM was safe and effective, and associated with sustained improvements in HbA1c and lower risk of hypoglycaemia.

Treatment of infertility with hypogonadotropic hypogonadism: 10-year experience in Auckland, New Zealand.

Infertility in idiopathic or acquired hypogonadotropic hypogonadism (HH) was managed with exogenous gonadotropins and artificial reproduction as needed, in Auckland, New Zealand, from 2000 to 2010. Of eight men seeking conception, 2/2 with acquired HH but only 2/6 with congenital HH achieved clinical pregnancy with exogenous gonadotropins, whereas 12/14 women (86%) achieved one or more live births. Current gonadotropin treatment does not seem to be optimal for men with congenital HH.

Maternal dexamethasone and EEG hyperactivity in preterm fetal sheep.

Maternal treatment with synthetic corticosteroids such as dexamethasone (DEX)significantly reduces neonatal morbidity and mortality, but its effects on the fetal brain remain unclear. In this study we evaluated the effects of DEX on EEG activity in preterm fetal sheep. Ewes at 103 days gestation received two intramuscular injections of DEX (12 mg, n = 8) or saline vehicle (n = 7) 24 h apart. Fetal EEG activity was recorded from 6 h before until 120 h after the first injection (DEX-1). DEX-1 was associated with a marked transient rise in total EEG power, maximal at 12 h (P < 0.001), with a relative increase in delta and reduced theta, alpha and beta activity, resolving by 24 h. Continuous EEG records showed a shift to larger but less frequent transient waveforms (P < 0.001). Unexpectedly, evolving epileptiform activity, consistent with electrographic and clinical seizures, developed from 178 ± 44 min after DEX-1.Similar but smaller changes were seen after the second injection. Following the injections, total power returned to control values, but the proportion of alpha activity progressively increased vs. controls (P < 0.001), with reduced interburst interval duration and number (P < 0.001). No histological neural injury or microglial activation was seen. In summary, exposure to maternal dexamethasone was associated with dramatic, evolving low-frequency hyperactivity on fetal cortical EEG recordings, followed by sustained changes consistent with maturation of fetal sleep architecture. We postulate that these effects may contribute to improved neonatal outcomes.

Pitfalls in the quest of neuroprotectants for the perinatal brain.

Sick preterm and term newborns are highly vulnerable to neural injury, and thus there has been a major search for new, safe and efficacious neuroprotective interventions in recent decades. Preclinical studies are essential to select candidate drugs for clinical trials in humans. This article focuses on 'negative' preclinical studies, i.e. studies where significant differences cannot be detected. Such findings are critical to inform both clinical and preclinical investigators, but historically they have been difficult to publish. A significant amount of time and resources is lost when negative results or nonpromising therapeutics are replicated in separate laboratories because these negative results were not shared with the research community in an open and accessible format. In this article, we discuss approaches to strengthen conclusions from negative preclinical studies and, conversely, to reduce false-negative preclinical evaluations of potential therapeutic compounds. Without being exhaustive, we address three major issues in conducting and interpreting preclinical experiments, including: (a) the choice of animal models, (b) the experimental design, and (c) issues concerning statistical analyses of the experiments. This general introduction is followed by synopses of negative data obtained from studies of three potential therapeutics for perinatal brain injury: (1) the somatostatin analog octreotide, (2) an AMPA/kainate receptor antagonist, topiramate, and (3) a pyruvate derivative, ethyl pyruvate.

Initial growth deceleration during GnRH analogue therapy for precocious puberty.

OBJECTIVES: To compare the efficacy of goserelin and leuprolide on initial deceleration of growth and weight gain during the first 12 months of GnRH analogue treatment for precocious puberty. DESIGN: Retrospective cohort analysis. PATIENTS: Forty children with precocious puberty treated with either goserelin or leuprolide (33 females, mean age 7.3 and 7.7 years, respectively, at the start of treatment). MEASUREMENTS: The primary outcomes were baseline-to-6-months and 6-months-to-12-months change in height standard deviation score (SDS) and body mass index (BMI). Relative tall stature was calculated as the difference between height SDS and mid-parental height (MPH) SDS at baseline. RESULTS: Goserelin and leuprolide were associated with similar suppression of serum LH during the first 12 months of treatment (P = 0.62). Greater relative tall stature was strongly associated with more advanced bone age, greater BMI SDS and with greater reduction in height SDS in the first 6 months. Adjusted for relative tall stature, goserelin therapy was associated with significantly greater suppression of growth than leuprolide (P = 0.025) in the first 6 months of treatment, with no subsequent change in the second 6 months. A similar, significant increase in BMI was seen with both analogues. CONCLUSIONS: Both GnRH analogues were associated with effective biochemical suppression of puberty; however, goserelin was more effective at reducing linear growth during the first 6 months. Relative tall stature was a major determinant of the initial response to treatment.