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Antenatal hypoxia-ischaemia (HI) in preterm fetal sheep can trigger delayed evolution of severe, cystic white matter injury (WMI), in a similar timecourse to WMI in preterm infants. We therefore examined how severe hypoxia-ischaemia affects recovery of electroencephalographic (EEG) activity. Chronically instrumented preterm fetal sheep (0.7 gestation) received 25 min of complete umbilical cord occlusion (UCO, n = 9) or sham occlusion (controls, n = 9), and recovered for 21 days. HI was associated with a shift to lower frequency EEG activity for the first 5 days with persisting loss of EEG power in the delta and theta bands, and initial loss of power in the alpha and beta bands in the first 14 days of recovery. In the final 3 days of recovery, there was a marked rhythmic shift towards higher frequency EEG activity after UCO. The UCO group spent less time in high-voltage sleep, and in the early evening (7:02 pm ± 47 min) abruptly stopped cycling between sleep states, with a shift to a high frequency state for 2 h 48 min ± 40 min, with tonic electromyographic activity. These findings demonstrate persisting EEG and sleep state dysmaturation after severe hypoxia-ischaemia. Loss of fetal or neonatal sleep state cycling in the early evening may be a useful biomarker for evolving cystic WMI.

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Hypoxia-ischaemia (HI) before birth is a key risk factor for stillbirth and severe neurodevelopmental disability in survivors, including cerebral palsy, although there are no reliable biomarkers to detect at risk fetuses that may have suffered a transient period of severe HI. We investigated time and frequency domain measures of fetal heart rate variability (FHRV) for 3 weeks after HI in preterm fetal sheep at 0.7 gestation (equivalent to preterm humans) until 0.8 gestation (equivalent to term humans). We have previously shown that this is associated with delayed development of severe white and grey matter injury, including cystic white matter injury (WMI) resembling that observed in human preterm infants. HI was associated with suppression of time and frequency domain measures of FHRV and reduced their circadian rhythmicity during the first 3 days of recovery. By contrast, circadian rhythms of multiple measures of FHRV were exaggerated over the final 2 weeks of recovery, mediated by a greater reduction in FHRV during the morning nadir, but no change in the evening peak. These data suggest that the time of day at which FHRV measurements are taken affects their diagnostic utility. We further propose that circadian changes in FHRV may be a low-cost, easily applied biomarker of antenatal HI and evolving brain injury. KEY POINTS: Hypoxia-ischaemia (HI) before birth is a key risk factor for stillbirth and probably for disability in survivors, although there are no reliable biomarkers for antenatal brain injury. In preterm fetal sheep, acute HI that is known to lead to delayed development of severe white and grey matter injury over 3 weeks, was associated with early suppression of multiple time and frequency domain measures of fetal heart rate variability (FHRV) and loss of their circadian rhythms during the first 3 days after HI. Over the final 2 weeks of recovery after HI, exaggerated circadian rhythms of frequency domain FHRV measures were observed. The morning nadirs were lower with no change in the evening peak of FHRV. Circadian changes in FHRV may be a low-cost, easily applied biomarker of antenatal HI and evolving brain injury.

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Brief repeated fetal hypoxaemia during labour can trigger intrapartum decelerations of the fetal heart rate (FHR) via the peripheral chemoreflex or the direct effects of myocardial hypoxia, but the relative contribution of these two mechanisms and how this balance changes with evolving fetal compromise remain unknown. In the present study, chronically instrumented near-term fetal sheep received surgical vagotomy (n = 8) or sham vagotomy (control, n = 11) to disable the peripheral chemoreflex and unmask myocardial hypoxia. One-minute complete umbilical cord occlusions (UCOs) were performed every 2.5 min for 4 h or until arterial pressure fell below 20 mmHg. Hypotension and severe acidaemia developed progressively after 65.7 ± 7.2 UCOs in control fetuses and 49.5 ± 7.8 UCOs after vagotomy. Vagotomy was associated with faster development of metabolic acidaemia and faster impairment of arterial pressure during UCOs without impairing centralization of blood flow or neurophysiological adaptation to UCOs. During the first half of the UCO series, before severe hypotension developed, vagotomy was associated with a marked increase in FHR during UCOs. After the onset of evolving severe hypotension, FHR fell faster in control fetuses during the first 20 s of UCOs, but FHR during the final 40 s of UCOs became progressively more similar between groups, with no difference in the nadir of decelerations. In conclusion, FHR decelerations were initiated and sustained by the peripheral chemoreflex at a time when fetuses were able to maintain arterial pressure. After the onset of evolving hypotension and acidaemia, the peripheral chemoreflex continued to initiate decelerations, but myocardial hypoxia became progressively more important in sustaining and deepening decelerations. KEY POINTS: Brief repeated hypoxaemia during labour can trigger fetal heart rate decelerations by either the peripheral chemoreflex or myocardial hypoxia, but how this balance changes with fetal compromise is unknown. Reflex control of fetal heart rate was disabled by vagotomy to unmask the effects of myocardial hypoxia in chronically instrumented fetal sheep. Fetuses were then subjected to repeated brief hypoxaemia consistent with the rates of uterine contractions during labour. We show that the peripheral chemoreflex controls brief decelerations in their entirety at a time when fetuses were able to maintain normal or increased arterial pressure. The peripheral chemoreflex still initiated decelerations even after the onset of evolving hypotension and acidaemia, but myocardial hypoxia made an increasing contribution to sustain and deepen decelerations.

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Reduced grey matter volume in preterm infants is associated with later disability, but its time course and relationship with white matter injury are not well understood. We recently showed that moderate-severe hypoxia-ischaemia (HI) in preterm fetal sheep led to severe cystic injury 2-3 weeks later. In the same cohort we now show profound hippocampal neuronal loss from 3 days after HI. By contrast, reduction in cortical area and perimeter developed much more slowly, with maximum reduction at day 21. There was transient upregulation of cleaved caspase-3-positive apoptosis in the cortex at day 3 but no change in neuronal density or macroscopic injury of the cortex. Both microglia and astrocytes were transiently upregulated in the grey matter. EEG power was initially profoundly suppressed but partially recovered by 21 days of recovery, and final power was correlated with white matter area (p < 0.001, r2 = 0.75, F = 24.19), cortical area (p = 0.004, r2 = 0.44, F = 11.90) and hippocampi area (p = 0.049, r2 = 0.23, F = 4.58). In conclusion, the present study suggests that in preterm fetal sheep, hippocampal injury is established within a few days of acute HI, but impaired cortical growth develops slowly, in a similar time course to severe white matter injury.

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Cystic white matter injury is highly associated with severe neurodevelopmental disability and cerebral palsy in preterm infants, yet its pathogenesis remains poorly understood and there is no established treatment. In the present study, we tested the hypothesis that slowly evolving cystic white matter injury after hypoxia-ischaemia is mediated by programmed necrosis initiated by tumour necrosis factor. Tumour necrosis factor blockade was begun 3 days after hypoxia-ischaemia to target the tertiary phase of injury, when most secondary cell death is thought to be complete. Chronically instrumented preterm foetal sheep (0.7 gestation) received 25 min of hypoxia-ischaemia induced by complete umbilical cord occlusion or sham-umbilical cord occlusion (controls, n = 10), followed by intracerebroventricular infusion of the soluble TNF inhibitor, Etanercept, at 3, 8 and 13 days after umbilical cord occlusion (n = 9) or vehicle (n = 9). Foetal brains were processed for histology at 21 days after umbilical cord occlusion. Umbilical cord occlusion with vehicle was associated with a spectrum of macroscopic white matter degeneration, including white matter atrophy, ventriculomegaly and overt temporal lobe cystic white matter injury. Oligodendrocyte maturational arrest and impaired labelling of myelin proteins, characteristic of diffuse white matter injury, was observed in the parietal lobe and surrounding the cystic lesions in the temporal lobe. Etanercept markedly attenuated cystic white matter injury on the side of the intracerebroventricular infusion, with partial contralateral protection. Further, Etanercept improved oligodendrocyte maturation and labelling of myelin proteins in the temporal and parietal lobes. The present study shows that cystic white matter injury reflects late-onset tertiary cell death mediated by delayed neuroinflammation through the tumour necrosis factor pathway. Delayed tumour necrosis factor blockade markedly attenuated cystic white matter injury and restored oligodendrocyte maturation and deficits in myelin protein expression. These data suggest that delayed tumour necrosis factor blockade may represent a viable therapeutic strategy to reduce the risk of cystic and diffuse white matter injury and potentially cerebral palsy after preterm birth, with a surprisingly wide therapeutic window.

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Perinatal hypoxia-ischemia (HI) is still a significant contributor to mortality and adverse neurodevelopmental outcomes in term and preterm infants. HI brain injury evolves over hours to days, and involves complex interactions between the endogenous protective and pathological processes. Understanding the timing of evolution of injury is vital to guide treatment. Post-HI recovery is associated with a typical neurophysiological profile, with stereotypic changes in cerebral perfusion and oxygenation. After the initial recovery, there is a delayed, prolonged reduction in cerebral perfusion related to metabolic suppression, followed by secondary deterioration with hyperperfusion and increased cerebral oxygenation, associated with altered neurovascular coupling and impaired cerebral autoregulation. These changes in cerebral perfusion are associated with the stages of evolution of injury and injury severity. Further, iatrogenic factors can also affect cerebral oxygenation during the early period of deranged metabolism, and improving clinical management may improve neuroprotection. We will review recent evidence that changes in cerebral oxygenation and metabolism after HI may be useful biomarkers of prognosis.

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Preterm birth continues to be associated with neurodevelopmental problems including cerebral palsy. Cystic white matter injury (WMI) is still the major neuropathology underlying cerebral palsy, affecting 1-3% of preterm infants. Although rates have gradually fallen over time, the pathogenesis and evolution of cystic WMI are still poorly understood. Hypoxia-ischemia (HI) remains an important contributor, yet there is no established treatment to prevent injury. Clinically, serial ultrasound and magnetic resonance imaging studies typically show delayed development of cystic lesions 2-4 weeks after birth. This raises the important and unresolved question as to whether this represents slow evolution of injury occurring around the time of birth or repeated injury over many weeks after birth. There is increasing evidence that tertiary injury after HI can contribute to impairment of white and grey matter maturation. In the present review, we discuss preclinical evidence that severe, cystic WMI can evolve for many weeks after acute HI and is associated with microglia activity. This suggests the intriguing hypothesis that the tertiary phase of injury is not as subtle as often thought and that there may be a window of therapeutic opportunity for 1 to 2 weeks after hypoxic-ischemic injury to prevent delayed cystic lesions, and so, further reduce the risk of cerebral palsy after preterm birth.

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White matter injury, including both diffuse and cystic elements, remains highly associated with neurodevelopmental disability and cerebral palsy in preterm infants, yet its pathogenesis and evolution are still poorly understood and there is no established treatment. We examined the long-term evolution of white matter injury in chronically instrumented preterm fetal sheep (0.7 gestation) after 25 min of complete umbilical cord occlusion or sham occlusion. Fetal brains were processed for histology after 3 days (n = 9, sham n = 9), 7 days (n = 8, sham n = 8), 14 days (n = 9, sham n = 8) and 21 days (n = 9, sham n = 9) of recovery. At 3 and 7 days recovery, umbilical cord occlusion was associated with diffuse white matter injury, with loss of total and mature oligodendrocytes and reduced myelination in both the parietal and temporal lobes. At 14 days after umbilical cord occlusion, extensive microglial and astrocytic activation were observed in the temporal lobe. At 21 days recovery a spectrum of severe white matter degeneration was observed, including white matter atrophy, ventriculomegaly and overt cystic white matter lesions. The most severe injury was observed in the temporal lobe after 21 days recovery, including the majority of cystic lesions, persistent oligodendrocyte maturational arrest and impaired myelination. The spatial distribution of delayed white matter degeneration at 21 days recovery was closely related to the location of dense microglial aggregates at earlier time-points, implicating a role for exuberant inflammation originating from microglial aggregates in the pathogenesis of cystic white matter injury. The delayed appearance of cystic injury is consistent with continuing tertiary evolution of necrotic cell death. This slow evolution raises the tantalizing possibility that there may a relatively long therapeutic window to mitigate the development of cystic white matter injury. Delayed anti-inflammatory treatments may therefore represent a promising strategy to reduce neurodevelopmental disability in the preterm infants.

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Impaired cardiac preload secondary to umbilical cord occlusion (UCO) has been hypothesized to contribute to intrapartum decelerations, brief falls in fetal heart rate (FHR), through activation of the Bezold-Jarisch reflex. This cardioprotective reflex increases parasympathetic and inhibits sympathetic outflows triggering hypotension, bradycardia, and peripheral vasodilation, but its potential to contribute to intrapartum decelerations has never been systematically examined. In this study, we performed bilateral cervical vagotomy to remove the afferent arm and the efferent parasympathetic arm of the Bezold-Jarisch reflex. Twenty-two chronically instrumented fetal sheep at 0.85 of gestation received vagotomy (n = 7) or sham vagotomy (control, n = 15), followed by three 1-min complete UCOs separated by 4-min reperfusion periods. UCOs in control fetuses were associated with a rapid fall in FHR and reduced femoral blood flow mediated by intense femoral vasoconstriction, leading to hypertension. Vagotomy abolished the rapid fall in FHR (P < 0.001) and, despite reduced diastolic filling time, increased both carotid (P < 0.001) and femoral (P < 0.05) blood flow during UCOs, secondary to carotid vasodilation (P < 0.01) and delayed femoral vasoconstriction (P < 0.05). Finally, vagotomy was associated with an attenuated rise in cortical impedance during UCOs (P < 0.05), consistent with improved cerebral substrate supply. In conclusion, increased carotid and femoral blood flows after vagotomy are consistent with increased left and right ventricular output, which is incompatible with the hypothesis that labor-like UCOs impair ventricular filling. Overall, the cardiovascular responses to vagotomy do not support the hypothesis that the Bezold-Jarisch reflex is activated by UCO. The Bezold-Jarisch reflex is therefore mechanistically unable to contribute to intrapartum decelerations.

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Antenatal glucocorticoids improve outcomes among premature infants but are associated with hyperglycemia, which can exacerbate hypoxic-ischemic injury. It is still unclear how antenatal glucocorticoids or hyperglycemia modulate fetal cardiovascular adaptations to severe asphyxia. In this study, preterm fetal sheep received either saline or 12 mg im maternal dexamethasone, followed 4 h later by complete umbilical cord occlusion (UCO) for 25 min. An additional cohort of fetuses received titrated glucose infusions followed 4 h later by UCO to control for the possibility that hyperglycemia contributed to the cardiovascular effects of dexamethasone. Fetuses were studied for 7 days after UCO. Maternal dexamethasone was associated with fetal hyperglycemia (P < 0.001), increased arterial pressure (P < 0.001), and reduced femoral (P < 0.005) and carotid (P < 0.05) vascular conductance before UCO. UCO was associated with bradycardia, femoral vasoconstriction, and transient hypertension. For the first 5 min of UCO, fetal blood pressure in the dexamethasone-asphyxia group was greater than saline-asphyxia (P < 0.001). However, the relative increase in arterial pressure was not different from saline-asphyxia. Fetal heart rate and femoral vascular conductance fell to similar nadirs in both saline and dexamethasone-asphyxia groups. Dexamethasone did not affect the progressive decline in femoral vascular tone or arterial pressure during continuing UCO. By contrast, there were no effects of glucose infusions on the response to UCO. In summary, maternal dexamethasone but not fetal hyperglycemia increased fetal arterial pressure before and for the first 5 min of prolonged UCO but did not augment the cardiovascular adaptations to acute asphyxia.

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