Tumour necrosis factor blockade after asphyxia in foetal sheep ameliorates cystic white matter injury.

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.

Circadian patterns of heart rate variability in fetal sheep after hypoxia-ischaemia: A biomarker of evolving brain injury.

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.

Magnesium sulphate reduces tertiary gliosis but does not improve EEG recovery or white or grey matter cell survival after asphyxia in preterm fetal sheep.

Maternal magnesium sulphate (MgSO4 ) treatment is widely recommended before preterm birth for neuroprotection. However, this is controversial because there is limited evidence that MgSO4 provides long-term neuroprotection. Preterm fetal sheep (104 days gestation; term is 147 days) were assigned randomly to receive sham occlusion with saline infusion (n = 6) or i.v. infusion with MgSO4 (n = 7) or vehicle (saline, n = 6) from 24 h before hypoxia-ischaemia induced by umbilical cord occlusion until 24 h after occlusion. Sheep were killed after 21 days of recovery, for fetal brain histology. Functionally, MgSO4 did not improve long-term EEG recovery. Histologically, in the premotor cortex and striatum, MgSO4 infusion attenuated post-occlusion astrocytosis (GFAP+ ) and microgliosis but did not affect numbers of amoeboid microglia or improve neuronal survival. In the periventricular and intragyral white matter, MgSO4 was associated with fewer total (Olig-2+ ) oligodendrocytes compared with vehicle + occlusion. Numbers of mature (CC1+ ) oligodendrocytes were reduced to a similar extent in both occlusion groups compared with sham occlusion. In contrast, MgSO4 was associated with an intermediate improvement in myelin density in the intragyral and periventricular white matter tracts. In conclusion, a clinically comparable dose of MgSO4 was associated with moderate improvements in white and grey matter gliosis and myelin density but did not improve EEG maturation or neuronal or oligodendrocyte survival. KEY POINTS: Magnesium sulphate is widely recommended before preterm birth for neuroprotection; however, there is limited evidence that magnesium sulphate provides long-term neuroprotection. In preterm fetal sheep exposed to hypoxia-ischaemia (HI), MgSO4 was associated with attenuated astrocytosis and microgliosis in the premotor cortex and striatum but did not improve neuronal survival after recovery to term-equivalent age, 21 days after HI. Magnesium sulphate was associated with loss of total oligodendrocytes in the periventricular and intragyral white matter tracts, whereas mature, myelinating oligodendrocytes were reduced to a similar extent in both occlusion groups. In the same regions, MgSO4 was associated with an intermediate improvement in myelin density. Functionally, MgSO4 did not improve long-term recovery of EEG power, frequency or sleep stage cycling. A clinically comparable dose of MgSO4 was associated with moderate improvements in white and grey matter gliosis and myelin density but did not improve EEG maturation or neuronal or oligodendrocyte survival.

Dissecting the contributions of the peripheral chemoreflex and myocardial hypoxia to fetal heart rate decelerations in near-term fetal sheep.

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.

Fetal defenses against intrapartum head compression-implications for intrapartum decelerations and hypoxic-ischemic injury.

Uterine contractions during labor and engagement of the fetus in the birth canal can compress the fetal head. Its impact on the fetus is unclear and still controversial. In this integrative physiological review, we highlight evidence that decelerations are uncommonly associated with fetal head compression. Next, the fetus has an impressive ability to adapt to increased intracranial pressure through activation of the intracranial baroreflex, such that fetal cerebral perfusion is well-maintained during labor, except in the setting of prolonged systemic hypoxemia leading to secondary cardiovascular compromise. Thus, when it occurs, fetal head compression is not necessarily benign but does not seem to be a common contributor to intrapartum decelerations. Finally, the intracranial baroreflex and the peripheral chemoreflex (the response to acute hypoxemia) have overlapping efferent effects. We propose the hypothesis that these reflexes may work synergistically to promote fetal adaptation to labor.

Physiological control of fetal heart rate variability during labour: implications and controversies.

The interpretation of fetal heart rate (FHR) patterns is the only available method to continuously monitor fetal well-being during labour. One of the most important yet contentious aspects of the FHR pattern is changes in FHR variability (FHRV). Some clinical studies suggest that loss of FHRV during labour is a sign of fetal compromise so this is reflected in practice guidelines. Surprisingly, there is little systematic evidence to support this observation. In this review we methodically dissect the potential pathways controlling FHRV during labour-like hypoxaemia. Before labour, FHRV is controlled by the combined activity of the parasympathetic and sympathetic nervous systems, in part regulated by a complex interplay between fetal sleep state and behaviour. By contrast, preclinical studies using multiple autonomic blockades have now shown that sympathetic neural control of FHRV was potently suppressed between periods of labour-like hypoxaemia, and thus, that the parasympathetic system is the sole neural regulator of FHRV once FHR decelerations are present during labour. We further discuss the pattern of changes in FHRV during progressive fetal compromise and highlight potential biochemical, behavioural and clinical factors that may regulate parasympathetic-mediated FHRV during labour. Further studies are needed to investigate the regulators of parasympathetic activity to better understand the dynamic changes in FHRV and their true utility during labour.

Is Late Prevention of Cerebral Palsy in Extremely Preterm Infants Plausible?

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.

Increased variability of fetal heart rate during labour: a review of preclinical and clinical studies.

Increased fetal heart rate variability (FHRV) in intrapartum cardiotocographic recording has been variably defined and poorly understood, limiting its clinical utility. Both preclinical (animal) and clinical (human) evidence support that increased FHRV is observed in the early stage of intrapartum fetal hypoxaemia but can also be observed in a subset of fetuses during the preterminal stage of repeated hypoxaemia. This review of available evidence provides data and expert opinion on the pathophysiology of increased FHRV, its clinical significance and a stepwise approach regarding the management of this pattern, and propose recommendations for standardisation of related terminology.

Adverse neural effects of delayed, intermittent treatment with rEPO after asphyxia in preterm fetal sheep.

KEY POINTS: We have previously shown that high-dose constant infusion of recombinant human erythropoietin (rEPO) from 30 min to 72 h after asphyxia in preterm fetal sheep reduced histological injury and improved electrophysiological recovery. This study shows that a high-dose infusion of rEPO from 6 to 72 h after asphyxia did not improve EEG recovery, oligodendrocyte and neuronal survival at 1 week post-asphyxia. Of concern, intermittent rEPO boluses started 6 h after asphyxia were associated with impaired EEG recovery and bilateral cystic injury of temporal lobe intragyral white matter. Intermittent boluses of rEPO were associated with significantly increased cerebral vascular resistance and hypoperfusion, particularly after the first dose, but did not affect seizures, suggesting mismatch between perfusion and brain activity. ABSTRACT: Recombinant human erythropoietin (rEPO) is a promising treatment for hypoxic-ischaemic brain injury. Disappointingly, a large randomized controlled trial in preterm infants found that prophylactic, repeated high-dose rEPO boluses started within 24 h of birth did not improve neurodevelopmental outcomes. We examined whether initiation of a continuous infusion of rEPO at the end of the latent phase after hypoxic-ischaemia (HI) might improve outcomes compared with intermittent bolus injections. Chronically instrumented preterm (0.7 gestation) fetal sheep received sham asphyxia or asphyxia induced by complete umbilical cord occlusion for 25 min. Six hours after asphyxia, fetuses received either a continuous infusion of rEPO (loading dose 2000 IU, infusion at 520 IU/h) from 6 to 72 h post-asphyxia or intravenous saline or 5000 IU rEPO, with repeated doses every 48 h for 5 days. Continuous infusion of rEPO did not improve EEG recovery, oligodendrocyte and neuronal survival at 1 week post-asphyxia. By contrast, intermittent rEPO boluses were associated with impaired EEG recovery and bilateral cystic injury of temporal lobe intragyral white matter in 6/8 fetuses. These studies demonstrate for the first time that initiation of intermittent rEPO boluses 6 h after HI, at a dose comparable with recent clinical trials, exacerbated neural injury. These data reinforce the importance of early initiation of many potential neuroprotective therapies.

Lack of evidence for impaired preload or Bezold-Jarisch activation during brief umbilical cord occlusions in fetal sheep.

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.

Transient effects of forebrain ischemia on fetal heart rate variability in fetal sheep.

Fetal heart rate variability (FHRV) is a key index of antenatal and intrapartum fetal well-being. FHRV is well established to be mediated by both arms of the autonomic nervous system, but it remains unknown whether higher centers in the forebrain contribute to FHRV. We tested the hypothesis that selective forebrain ischemia would impair the generation of FHRV. Sixteen chronically instrumented near-term fetal sheep were subjected to either forebrain ischemia induced by bilateral carotid occlusion or sham-ischemia for 30 min. Time, frequency, and nonlinear measures of FHRV were assessed during and for seven days after ischemia. Ischemia was associated with profound suppression of electroencephalographic (EEG) power, which remained suppressed throughout the recovery period (P < 0.001). During the first 5 min of ischemia, multiple time and frequency domain measures were increased (all P < 0.05) before returning back to sham levels. A delayed increase in sample entropy was observed during ischemia (P < 0.05). For the first 3 h after ischemia, there was moderate suppression of two measures of FHRV (very-low frequency power and the standard deviation of RR-intervals, both P < 0.05) and increased sample entropy (P < 0.05). Thereafter, all measures of FHRV returned to control levels. In conclusion, profound forebrain ischemia sufficient to lead to severe neural injury had only transient effect on multiple measures of FHRV. These findings suggest that the forebrain makes a limited contribution to FHRV. FHRV therefore primarily originates in the hindbrain and is unlikely to provide meaningful information on forebrain neurodevelopment or metabolism.

Treating Seizures after Hypoxic-Ischemic Encephalopathy-Current Controversies and Future Directions.

Seizures are common in newborn infants with hypoxic-ischemic encephalopathy and are highly associated with adverse neurodevelopmental outcomes. The impact of seizure activity on the developing brain and the most effective way to manage these seizures remain surprisingly poorly understood, particularly in the era of therapeutic hypothermia. Critically, the extent to which seizures exacerbate brain injury or merely reflect the underlying evolution of injury is unclear. Current anticonvulsants, such as phenobarbital and phenytoin have poor efficacy and preclinical studies suggest that most anticonvulsants are associated with adverse effects on the developing brain. Levetiracetam seems to have less potential neurotoxic effects than other anticonvulsants but may not be more effective. Given that therapeutic hypothermia itself has significant anticonvulsant effects, randomized controlled trials of anticonvulsants combined with therapeutic hypothermia, are required to properly determine the safety and efficacy of these drugs. Small clinical studies suggest that prophylactic phenobarbital administration may improve neurodevelopmental outcomes compared to delayed administration; however, larger high-quality studies are required to confirm this. In conclusion, there is a distinct lack of high-quality evidence for whether and to what extent neonatal seizures exacerbate brain damage after hypoxia-ischemia and how best to manage them in the era of therapeutic hypothermia.

Peripheral chemoreflex control of fetal heart rate decelerations overwhelms the baroreflex during brief umbilical cord occlusions in fetal sheep.

KEY POINTS: The majority of intrapartum decelerations are widely believed to be mediated by the baroreflex secondary to brief umbilical cord occlusions (UCOs) but this remains unproven. We examined the responses to brief-UCOs in fetal sheep and compared these to a phenylephrine-stimulated baroreflex in a separate cohort. A further cohort was instrumented with near-infrared spectroscopy to measure cerebral oxygenation during UCO. The first 3-4 s of the brief-UCOs were consistent with a baroreflex, and associated with a minor fall in fetal heart rate (FHR). Thereafter, the remainder of the FHR decelerations were highly consistent with the peripheral chemoreflex. The baroreflex is not sufficient to produce deep, rapid decelerations characteristic of variable decelerations and it is therefore likely to be a minor contributor to intrapartum decelerations. ABSTRACT: Fetal heart rate (FHR) monitoring is widely used to assess fetal wellbeing during labour, yet the physiology underlying FHR patterns remains incompletely understood. The baroreflex is widely believed to mediate brief intrapartum decelerations, but evidence supporting this theory is lacking. We therefore investigated the physiological changes in near-term fetal sheep during brief repeated umbilical cord occlusions (brief-UCOs, n = 15). We compared this to separate cohorts that underwent a phenylephrine challenge to stimulate the baroreflex (n = 9) or were instrumented with near-infrared spectroscopy and underwent prolonged 15-min complete UCO (prolonged-UCO, n = 9). The first 3-4 s of brief-UCOs were associated with hypertension (P = 0.000), a fall in FHR by 9.7-16.9 bpm (P = 0.002). The FHR/MAP relationship during this time was consistent with that observed during a phenylephrine-induced baroreflex. At 4-5 s, the FHR/MAP relationship began to deviate from the phenylephrine baroreflex curve as FHR fell independently of MAP until its nadir in association with intense peripheral vasoconstriction (P = 0.000). During prolonged-UCO, cerebral oxygenation remained steady until 4 s after the start of prolonged-UCO, and then began to fall (P = 0.000). FHR and cerebral oxygenation then fell in parallel until the FHR nadir. In conclusion, the baroreflex has a minor role in mediating the first 3-4 s of FHR decelerations during complete UCO, but thereafter the peripheral chemoreflex is the dominant mediator. Overall, the baroreflex is neither necessary nor sufficient to produce deep, rapid decelerations characteristic of variable decelerations; it is therefore likely to be a minor contributor to intrapartum decelerations.

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.

Lipopolysaccharide-induced changes in the neurovascular unit in the preterm fetal sheep brain.

BACKGROUND: Exposure to inflammation during pregnancy can predispose to brain injury in premature infants. In the present study, we investigated the effects of prolonged exposure to inflammation on the cerebrovasculature of preterm fetal sheep. METHODS: Chronically instrumented fetal sheep at 103-104 days of gestation (full term is ~ 147 days) received continuous low-dose lipopolysaccharide (LPS) infusions (100 ng/kg over 24 h, followed by 250 ng/kg/24 h for 96 h plus boluses of 1 µg LPS at 48, 72, and 96 h) or the same volume of normal saline (0.9%, w/v). Ten days after the start of LPS exposure at 113-114 days of gestation, the sheep were killed, and the fetal brain perfused with formalin in situ. Vessel density, pericyte and astrocyte coverage of the blood vessels, and astrogliosis in the cerebral cortex and white matter were determined using immunohistochemistry. RESULTS: LPS exposure reduced (P < 0.05) microvascular vessel density and pericyte vascular coverage in the cerebral cortex and white matter of preterm fetal sheep, and increased the activation of perivascular astrocytes, but decreased astrocytic vessel coverage in the white matter. CONCLUSIONS: Prolonged exposure to LPS in preterm fetal sheep resulted in decreased vessel density and neurovascular remodeling, suggesting that chronic inflammation adversely affects the neurovascular unit and, therefore, could contribute to long-term impairment of brain development.

Effects of ß-adrenergic stimulation on fetal heart rate, heart rate variability, and T-wave elevation during brief umbilical cord occlusions in fetal sheep.

Circulating catecholamines are critical for fetal adaptation to hypoxia by regulating fetal heart rate (FHR) and promoting myocardial contractility and peripheral vasoconstriction. They have been hypothesized to contribute to changes in FHR variability (FHRV) and T-wave morphology, clinical indexes of fetal well-being during labor. ß-Adrenergic blockade with propranolol does not affect FHRV during labor-like hypoxemia and only attenuated the increase in T-wave height between the episodes of hypoxemia. To further investigate the potential role of catecholamines, we investigated whether pharmacological ß-adrenergic stimulation could increase FHRV and T-wave elevation during intermittent labor-like hypoxemia. Nineteen chronically instrumented fetal sheep at 0.85 of gestation received isoprenaline hydrochloride (n = 7) or saline (control, n = 12), followed by three 1-min complete umbilical cord occlusions (UCOs) separated by 4-min reperfusion periods. Before the UCOs, infusion of isoprenaline increased FHR (P < 0.001), absolute-T/QRS ratio (P < 0.001), and one measure of FHRV [root-mean-square of successive RR interval differences (RMSSD), P < 0.05]. UCOs triggered deep FHR decelerations. During UCOs, isoprenaline was associated with increased FHR (P < 0.001) and absolute-T/QRS ratio (P < 0.05), but no effect on T/QRS ratio was observed when normalized to baseline before UCOs (normalized-T/QRS ratio). Between UCOs, isoprenaline increased FHR (P < 0.001) and absolute-T/QRS ratio (P < 0.05) but did not affect normalized-T/QRS ratio or any measures of FHRV. Arterial pressure was not affected by isoprenaline at any point. Our findings indicate that circulating catecholamines regulate FHR but not FHRV during labor-like hypoxemia and promote T-wave elevation between but not during intermittent fetal hypoxemia.

Parasympathetic activity is the key regulator of heart rate variability between decelerations during brief repeated umbilical cord occlusions in fetal sheep.

Fetal heart rate variability (FHRV) is a widely used index of intrapartum well being. Both arms of the autonomic system regulate FHRV under normoxic conditions in the antenatal period. However, autonomic control of FHRV during labor when the fetus is exposed to repeated, brief hypoxemia during uterine contractions is poorly understood. We have previously shown that the sympathetic nervous system (SNS) does not regulate FHRV during labor-like hypoxia. We therefore investigated the hypothesis that the parasympathetic system is the main mediator of intrapartum FHRV. Twenty-six chronically instrumented fetal sheep at 0.85 of gestation received either bilateral cervical vagotomy (n = 7), atropine sulfate (n = 7), or sham treatment (control, n = 12), followed by three 1-min complete umbilical cord occlusions (UCOs) separated by 4-min reperfusion periods. Parasympathetic blockade reduced three measures of FHRV before UCOs (all P < 0.01). Between UCOs, atropine and vagotomy were associated with marked tachycardia (both P < 0.005), suppressed measures of FHRV (all P < 0.01), and abolished FHRV on visual inspection compared with the control group. Tachycardia in the atropine and vagotomy groups resolved over the first 10 min after the final UCO, in association with evidence that the SNS contribution to FHRV progressively returned during this time. Our findings support that SNS control of FHRV is acutely suppressed for at least 4 min after a deep intrapartum deceleration and takes 5-10 min to recover. The parasympathetic system is therefore likely to be the key mediator of FHRV once frequent FHR decelerations are established during labor.

Effects of antenatal dexamethasone and hyperglycemia on cardiovascular adaptation to asphyxia in preterm fetal sheep.

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.

Circulating catecholamines partially regulate T-wave morphology but not heart rate variability during repeated umbilical cord occlusions in fetal sheep.

Fetal heart rate (FHR) variability (FHRV) and ST segment morphology are potential clinical indices of fetal well-being during labor. ß-Adrenergic stimulation by circulating catecholamines has been hypothesized to contribute to both FHRV and ST segment morphology during labor, but this has not been tested during brief repeated fetal hypoxemia that is characteristic of labor. Near-term fetal sheep (0.85 gestation) received propranolol (ß-adrenergic blockade; n = 10) or saline (n = 7) 30 min before being exposed to three 2-min complete umbilical cord occlusions (UCOs) separated by 3-min reperfusions. T/QRS ratio was calculated throughout UCOs and reperfusion periods, and measures of FHRV (RMSSD, SDNN, and STV) were calculated between UCOs. During the baseline period, before the start of UCOs, propranolol was associated with reduced FHR, SDNN, and STV but did not affect RMSSD or T/QRS ratio. UCOs were associated with rapid FHR decelerations and increased T/QRS ratio; propranolol significantly reduced FHR during UCOs and was associated with a slower rise in T/QRS ratio during the first UCOs, without affecting the maximal rise or T/QRS ratio during the second and third UCO. Between UCOs propranolol reduced FHR and T/QRS ratio but did not affect any measure of FHRV. These data demonstrate that circulating catecholamines do not contribute to FHRV during labor-like hypoxemia. Furthermore, circulating catecholamines did not contribute to the major rise in T/QRS ratio during labor-like hypoxemia but may regulate T/QRS ratio between brief hypoxemia.

Plasma vasopressin levels are closely associated with fetal hypotension and neuronal injury after hypoxia-ischemia in near-term fetal sheep.

BACKGROUND: Sensitive biomarkers are needed to rapidly identify high-risk infants after hypoxia-ischemia for neuroprotective treatment. Hypotension is a key determinant of hypoxic-ischemic neural injury, and a potent stimulus of humoral pressors including angiotensin-II and arginine vasopressin. We therefore aimed to quantify the relationship between vasopressin and angiotensin-II levels in the latent phase after hypoxia-ischemia induced by umbilical cord occlusion (UCO) with both the severity of preceding hypotension and subsequent neuronal injury. METHODS: Chronically instrumented near-term fetal sheep underwent sham-UCO or UCO for either 15 min or until mean arterial pressure was <8 mmHg. Neuronal injury was assessed after 72 h recovery. RESULTS: Umbilical cord occlusion was associated with severe hypotension that recovered after UCO; two fetuses developed profound secondary hypotension within 6 h and died. Vasopressin levels but not angiotensin-II were significantly elevated 1-3 h after UCO and were closely associated with the severity of hypotension during UCO and the subsequent severity of neuronal loss in the parasagittal and lateral cortex, caudate nucleus and putamen. The Youden cut-point for vasopressin at 1 h was 180.0 pmol/L, with sensitivity 100% and specificity 92.3% for severe neuronal injury or death. CONCLUSION: Vasopressin levels shortly after moderate-severe hypoxia-ischemia may be a useful early biomarker to guide the timely implementation of neuroprotective treatment. IMPACT: It can be difficuIt to rapidly identify infants who might benefit from therapeutic hypothermia. We investigated whether increases in plasma pressor hormones early after hypoxia-ischemia were biomarkers for neonatal hypoxic-ischemic encephalopathy using near-term fetal sheep. Arginine vasopressin levels were elevated at 1-3 h after hypoxia-ischemia and were predictive of the severity of preceding hypotension and subsequent risk of severe neuronal injury or death after hypoxia-ischemia. Arginine vasopressin may help identify neonates at high risk of hypoxic-ischemic encephalopathy early within the therapeutic window for hypothermia.