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.

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.

Perinatal brain injury: mechanisms and therapeutic approaches.

Brain damage resulting from perinatal hypoxia-ischemia evolves slowly over time. While a small number of brain cells may die during a sufficiently profound period of hypoxia-ischemia, many will show initial recovery during a "latent" phase characterized by actively suppressed neural metabolism and activity. Critically, this transient recovery may be followed after ~6 hours by a phase of secondary deterioration, with delayed seizures, failure of mitochondrial function, cytotoxic edema, and bulk cell death over ~72 hours. This is followed by a tertiary phase of remodeling and recovery. Understanding the mechanisms of injury that occur during each phase may allow for the development of more targeted treatments. This review discusses the mechanisms of injury that occur during the primary, latent, secondary and tertiary phases of injury and potential treatments that target one or more of these phases. Treatment during the latent phase has the greatest potential to prevent injury. In the secondary phase of injury, anticonvulsants can attenuate seizures but show limited neuroprotection. By contrast, there is increasing preclinical evidence that neurorestorative therapies may improve long-term outcomes.

Chronic inflammation and impaired development of the preterm brain.

The preterm newborn is at significant risk of neural injury and impaired neurodevelopment. Infants with mild or no evidence of injury may also be at risk of altered brain development, with evidence impaired cell maturation. The underlying causes are multifactorial and include exposure of both the fetus and newborn to hypoxia-ischemia, inflammation (chorioamnionitis) and infection, adverse maternal lifestyle choices (smoking, drug and alcohol use, diet) and obesity, as well as the significant demand that adaptation to post-natal life places on immature organs. Further, many fetuses and infants may have combinations of these events, and repeated (multi-hit) events that may induce tolerance to injury or sensitize to greater injury. Currently there are no treatments to prevent preterm injury or impaired neurodevelopment. However, inflammation is a common pathway for many of these insults, and clinical and experimental evidence demonstrates that acute and chronic inflammation is associated with impaired brain development. This review examines our current knowledge about the relationship between inflammation and preterm brain development, and the potential for stem cell therapy to provide neuroprotection and neurorepair through reducing inflammation and release of trophic factors, which promote cell maturation and repair.

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.

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.

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.

Antenatal dexamethasone after asphyxia increases neural injury in preterm fetal sheep.

BACKGROUND AND PURPOSE: Maternal glucocorticoid treatment for threatened premature delivery dramatically improves neonatal survival and short-term morbidity; however, its effects on neurodevelopmental outcome are variable. We investigated the effect of maternal glucocorticoid exposure after acute asphyxia on injury in the preterm brain. METHODS: Chronically instrumented singleton fetal sheep at 0.7 of gestation received asphyxia induced by complete umbilical cord occlusion for 25 minutes. 15 minutes after release of occlusion, ewes received a 3 ml i.m. injection of either dexamethasone (12 mg, n = 10) or saline (n = 10). Sheep were killed after 7 days recovery; survival of neurons in the hippocampus and basal ganglia, and oligodendrocytes in periventricular white matter were assessed using an unbiased stereological approach. RESULTS: Maternal dexamethasone after asphyxia was associated with more severe loss of neurons in the hippocampus (CA3 regions, 290 ± 76 vs 484 ± 98 neurons/mm(2), mean ± SEM, P<0.05) and basal ganglia (putamen, 538 ± 112 vs 814 ± 34 neurons/mm(2), P<0.05) compared to asphyxia-saline, and with greater loss of both total (913 ± 77 vs 1201 ± 75/mm(2), P<0.05) and immature/mature myelinating oligodendrocytes in periventricular white matter (66 ± 8 vs 114 ± 12/mm(2), P<0.05, vs sham controls 165 ± 10/mm(2), P<0.001). This was associated with transient hyperglycemia (peak 3.5 ± 0.2 vs. 1.4 ± 0.2 mmol/L at 6 h, P<0.05) and reduced suppression of EEG power in the first 24 h after occlusion (maximum -1.5 ± 1.2 dB vs. -5.0 ± 1.4 dB in saline controls, P<0.01), but later onset and fewer overt seizures. CONCLUSIONS: In preterm fetal sheep, exposure to maternal dexamethasone during recovery from asphyxia exacerbated brain damage.

Partial neuroprotection by nNOS inhibition during profound asphyxia in preterm fetal sheep.

Preterm brain injury is partly associated with hypoxia-ischemia starting before birth. Excessive nitric oxide production during HI may cause nitrosative stress, leading to cell membrane and mitochondrial damage. We therefore tested the hypothesis that therapy with a new, selective neuronal nitric oxide synthase (nNOS) inhibitor, JI-10 (0.022mg/kg bolus, n=8), given 30min before 25min of complete umbilical cord occlusion was protective in preterm fetal sheep at 101-104day gestation (term is 147days), compared to saline (n=8). JI-10 had no effect on fetal blood pressure, heart rate, carotid and femoral blood flow, total EEG power, nuchal activity, temperature or intracerebral oxygenation on near-infrared spectroscopy during or after occlusion. JI-10 was associated with later onset of post-asphyxial seizures compared with saline (p<0.05), and attenuation of the subsequent progressive loss of cytochrome oxidase (p<0.05). After 7days recovery, JI-10 was associated with improved neuronal survival in the caudate nucleus (p<0.05), but not the putamen or hippocampus, and more CNPase positive oligodendrocytes in the periventricular white matter (p<0.05). In conclusion, prophylactic nNOS inhibition before profound asphyxia was associated with delayed onset of seizures, slower decline of cytochrome oxidase and partial white and gray matter protection, consistent with protection of mitochondrial function.

Randomized controlled trial of a car safety seat insert to reduce hypoxia in term infants.

OBJECTIVE: To test the hypothesis that a foam plastic insert that allows the infant head to rest in a neutral position in sleep may prevent obstruction of the upper airway and thus reduce episodes of reduced oxygenation in term infants in car seats. METHODS: Healthy full-term babies were randomized to be studied during sleep while restrained in an infant car safety seat either with or without the insert, with continuous polysomnographic recordings with sleep video. RESULTS: Seventy-eight infants (39 in each group) had polysomnogram recordings at a mean of 8 days of age. Both groups showed a small fall in mean hemoglobin oxygen saturation (SpO2) over the first hour of sleep. There was no difference between insert and no insert in the rate of moderate desaturations (a fall in SpO2 ≥ 4% lasting for ≥ 10 seconds, mean ± SEM, 17.0 ± 1.5 vs 17.2 ± 1.5/hour), or mean SpO2 during sleep. The insert was associated with a significant reduction in the rate of obstructive apnea (0.3 ± 0.1 vs 0.9 ± 1.5/hour, P < .03), the severity of desaturation events (minimum SpO2 82% ± 1% vs 74% ± 2%, P < .001), and time with SpO2 <85% (0.6% ± 0.3% vs 1.8% ± 1.4%, P = .03). CONCLUSIONS: In full-term newborn infants, a car seat insert that helps the head to lie in a neutral position was associated with reduced severity of desaturation events but not the overall rate of moderate desaturations.

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.

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.

Glucocorticoids and preterm hypoxic-ischemic brain injury: the good and the bad.

Fetuses at risk of premature delivery are now routinely exposed to maternal treatment with synthetic glucocorticoids. In randomized clinical trials, these substantially reduce acute neonatal systemic morbidity, and mortality, after premature birth and reduce intraventricular hemorrhage. However, the overall neurodevelopmental impact is surprisingly unclear; worryingly, postnatal glucocorticoids are consistently associated with impaired brain development. We review the clinical and experimental evidence on how glucocorticoids may affect the developing brain and highlight the need for systematic research.

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.

Effect of cerebral hypothermia and asphyxia on the subventricular zone and white matter tracts in preterm fetal sheep.

Prolonged, moderate cerebral hypothermia is consistently neuroprotective after experimental hypoxia-ischemia. We have previously shown that hypothermia is also protective after profound asphyxia in the preterm brain. However, there is a concern whether hypothermia could suppress the proliferative response to injury in the white matter or subventricular zone (SVZ). Preterm (0.7 gestation) fetal sheep received complete umbilical cord occlusion for 25 min followed by cerebral hypothermia (extradural temperature reduced from 39.4±0.3 to 29.5±2.6°C) from 90 min to 70h after the end of occlusion or sham cooling. Occlusion-normothermia was associated with no effect on CNPase+ cells, but loss of O4+ oligodendrocytes, induction of cleaved caspase-3, and IB4+ microglia in the gyral and periventricular white matter compared to sham-occlusion (p < 0.05), with a significant increase in KI67+ cells in the periventricular white matter (p < 0.05). Hypothermia was associated with significant protection of O4+ cells, with suppression of IB4+ microglia and KI67+ cells in the periventricular white matter. There was no significant change in astrocytes, microglia, KI67+, or caspase-3+ cells in the SVZ after asphyxia. In conclusion, this study provides strong support for the selective vulnerability of immature oligodendrocytes to a highly relevant insult in the fetal sheep. Although white matter protection with cerebral hypothermia was associated with reduced proliferation in the white matter tracts, it did not impair proliferation in the SVZ.

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.