Slow rewarming after hypothermia does not ameliorate white matter injury after hypoxia-ischemia in near-term fetal sheep.

BACKGROUND: The optimal rate to rewarm infants after therapeutic hypothermia is unclear. In this study we examined whether slow rewarming after 72 h of hypothermia would attenuate white matter injury. METHODS: Near-term fetal sheep received sham occlusion (n = 8) or cerebral ischemia for 30 min, followed by normothermia (n = 7) or hypothermia from 3-72 h, with either spontaneous fast rewarming (n = 8) within 1 h, or slow rewarming at ~0.5 °C/h (n = 8) over 10 h. Fetuses were euthanized 7 days later. RESULTS: Ischemia was associated with loss of total and mature oligodendrocytes, reduced expression of myelin proteins and induction of microglia and astrocytes, compared with sham controls (P < 0.05). Both hypothermia protocols were associated with a significant increase in numbers of total and mature oligodendrocytes, area fraction of myelin proteins and reduced numbers of microglia and astrocytes, compared with ischemia-normothermia (P < 0.05). There was no difference in the number of oligodendrocytes, microglia or astrocytes or expression of myelin proteins between fast and slow rewarming after hypothermia. CONCLUSION: The rate of rewarming after a clinically relevant duration of hypothermia had no apparent effect on white matter protection by hypothermia after cerebral ischemia in near-term fetal sheep. IMPACT: Persistent white matter injury is a major contributor to long-term disability after neonatal encephalopathy despite treatment with therapeutic hypothermia. The optimal rate to rewarm infants after therapeutic hypothermia is unclear; current protocols were developed on a precautionary basis. We now show that slow rewarming at 0.5 °C/h did not improve histological white matter injury compared with rapid spontaneous rewarming after a clinically established duration of hypothermia in near-term fetal sheep.

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.

Therapeutic Hypothermia Attenuates Cortical Interneuron Loss after Cerebral Ischemia in Near-Term Fetal Sheep.

Therapeutic hypothermia significantly improves outcomes after neonatal hypoxic-ischemic (HI) encephalopathy but is only partially protective. There is evidence that cortical inhibitory interneuron circuits are particularly vulnerable to HI and that loss of interneurons may be an important contributor to long-term neurological dysfunction in these infants. In the present study, we examined the hypothesis that the duration of hypothermia has differential effects on interneuron survival after HI. Near-term fetal sheep received sham ischemia or cerebral ischemia for 30 min, followed by cerebral hypothermia from 3 h after ischemia end and continued up to 48 h, 72 h, or 120 h recovery. Sheep were euthanized after 7 days for histology. Hypothermia up to 48 h recovery resulted in moderate neuroprotection of glutamate decarboxylase (GAD)+ and parvalbumin+ interneurons but did not improve survival of calbindin+ cells. Hypothermia up to 72 h recovery was associated with significantly increased survival of all three interneuron phenotypes compared with sham controls. By contrast, while hypothermia up to 120 h recovery did not further improve (or impair) GAD+ or parvalbumin+ neuronal survival compared with hypothermia up to 72 h, it was associated with decreased survival of calbindin+ interneurons. Finally, protection of parvalbumin+ and GAD+ interneurons, but not calbindin+ interneurons, with hypothermia was associated with improved recovery of electroencephalographic (EEG) power and frequency by day 7 after HI. The present study demonstrates differential effects of increasing the duration of hypothermia on interneuron survival after HI in near-term fetal sheep. These findings may contribute to the apparent preclinical and clinical lack of benefit of very prolonged hypothermia.

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.

Persistent cortical and white matter inflammation after therapeutic hypothermia for ischemia in near-term fetal sheep.

BACKGROUND: Therapeutic hypothermia significantly improves outcomes after moderate-severe hypoxic-ischemic encephalopathy (HIE), but it is partially effective. Although hypothermia is consistently associated with reduced microgliosis, it is still unclear whether it normalizes microglial morphology and phenotype. METHODS: Near-term fetal sheep (n = 24) were randomized to sham control, ischemia-normothermia, or ischemia-hypothermia. Brain sections were immunohistochemically labeled to assess neurons, microglia and their interactions with neurons, astrocytes, myelination, and gitter cells (microglia with cytoplasmic lipid granules) 7 days after cerebral ischemia. Lesions were defined as areas with complete loss of cells. RNAscope® was used to assess microglial phenotype markers CD86 and CD206. RESULTS: Ischemia-normothermia was associated with severe loss of neurons and myelin (p < 0.05), with extensive lesions, astrogliosis and microgliosis with a high proportion of gitter cells (p < 0.05). Microglial wrapping of neurons was present in both the ischemia groups. Hypothermia improved neuronal survival, suppressed lesions, gitter cells and gliosis (p < 0.05), and attenuated the reduction of myelin area fraction. The "M1" marker CD86 and "M2" marker CD206 were upregulated after ischemia. Hypothermia partially suppressed CD86 in the cortex only (p < 0.05), but did not affect CD206. CONCLUSIONS: Hypothermia prevented lesions after cerebral ischemia, but only partially suppressed microglial wrapping and M1 marker expression. These data support the hypothesis that persistent upregulation of injurious microglial activity may contribute to partial neuroprotection after hypothermia, and that immunomodulation after rewarming may be an important therapeutic target.

Targeting Persistent Neuroinflammation after Hypoxic-Ischemic Encephalopathy-Is Exendin-4 the Answer?

Hypoxic-ischemic encephalopathy is brain injury resulting from the loss of oxygen and blood supply around the time of birth. It is associated with a high risk of death or disability. The only approved treatment is therapeutic hypothermia. Therapeutic hypothermia has consistently been shown to significantly reduce the risk of death and disability in infants with hypoxic-ischemic encephalopathy. However, approximately 29% of infants treated with therapeutic hypothermia still develop disability. Recent preclinical and clinical studies have shown that there is still persistent neuroinflammation even after treating with therapeutic hypothermia, which may contribute to the deficits seen in infants despite treatment. This suggests that potentially targeting this persistent neuroinflammation would have an additive benefit in addition to therapeutic hypothermia. A potential additive treatment is Exendin-4, which is a glucagon-like peptide 1 receptor agonist. Preclinical data from various in vitro and in vivo disease models have shown that Exendin-4 has anti-inflammatory, mitochondrial protective, anti-apoptotic, anti-oxidative and neurotrophic effects. Although preclinical studies of the effect of Exendin-4 in perinatal hypoxic-ischemic brain injury are limited, a seminal study in neonatal mice showed that Exendin-4 had promising neuroprotective effects. Further studies on Exendin-4 neuroprotection for perinatal hypoxic-ischemic brain injury, including in large animal translational models are warranted to better understand its safety, window of opportunity and effectiveness as an adjunct with therapeutic hypothermia.

Lack of Neuroprotection with a Single Intravenous Infusion of Human Amnion Epithelial Cells after Severe Hypoxia-Ischemia in Near-Term Fetal Sheep.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) around the time of birth results from loss of oxygen (hypoxia) and blood supply (ischemia). Exogenous infusion of multi-potential cells, including human amnion epithelial cells (hAECs), can reduce hypoxic-ischemic (HI) brain injury. However, there are few data on treatment of severe HI in large animal paradigms at term. The aim of the current study was to determine whether infusion of hAECs early after injury may reduce brain damage after ischemia in near-term fetal sheep. METHODS: Chronically instrumented fetal sheep (0.85 gestation) received 30 min of global cerebral ischemia followed by intravenous infusion of hAECs from 2 h after the end of ischemia (ischemia-hAEC, n = 6) or saline (ischemia-vehicle, n = 7). Sham control animals received sham ischemia with vehicle infusion (sham control, n = 8). RESULTS: Ischemia was associated with significant suppression of EEG power and spectral edge frequency until the end of the experiment and a secondary rise in cortical impedance from 24 to 72 h, which were not attenuated by hAEC administration. Ischemia was associated with loss of neurons in the cortex, thalamus, striatum and hippocampus, loss of white matter oligodendrocytes and increased microglial numbers in the white matter, which were not affected by hAEC infusion. CONCLUSIONS: A single intravenous administration of hAECs did not reduce electrographic or histological brain damage after 30 min of global cerebral ischemia in near-term fetal sheep.

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.

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.

Preventing Brain Injury in the Preterm Infant-Current Controversies and Potential Therapies.

Preterm birth is associated with a high risk of morbidity and mortality including brain damage and cerebral palsy. The development of brain injury in the preterm infant may be influenced by many factors including perinatal asphyxia, infection/inflammation, chronic hypoxia and exposure to treatments such as mechanical ventilation and corticosteroids. There are currently very limited treatment options available. In clinical trials, magnesium sulfate has been associated with a small, significant reduction in the risk of cerebral palsy and gross motor dysfunction in early childhood but no effect on the combined outcome of death or disability, and longer-term follow up to date has not shown improved neurological outcomes in school-age children. Recombinant erythropoietin has shown neuroprotective potential in preclinical studies but two large randomized trials, in extremely preterm infants, of treatment started within 24 or 48 h of birth showed no effect on the risk of severe neurodevelopmental impairment or death at 2 years of age. Preclinical studies have highlighted a number of promising neuroprotective treatments, such as therapeutic hypothermia, melatonin, human amnion epithelial cells, umbilical cord blood and vitamin D supplementation, which may be useful at reducing brain damage in preterm infants. Moreover, refinements of clinical care of preterm infants have the potential to influence later neurological outcomes, including the administration of antenatal and postnatal corticosteroids and more accurate identification and targeted treatment of seizures.

Changes in Cellular Localization of Inter-Alpha Inhibitor Proteins after Cerebral Ischemia in the Near-Term Ovine Fetus.

Inter-alpha Inhibitor Proteins (IAIPs) are key immunomodulatory molecules. Endogenous IAIPs are present in human, rodent, and sheep brains, and are variably localized to the cytoplasm and nuclei at multiple developmental stages. We have previously reported that ischemia-reperfusion (I/R) reduces IAIP concentrations in the fetal sheep brain. In this study, we examined the effect of I/R on total, cytoplasmic, and nuclear expression of IAIPs in neurons (NeuN+), microglia (Iba1+), oligodendrocytes (Olig2+) and proliferating cells (Ki67+), and their co-localization with histones and the endoplasmic reticulum in fetal brain cells. At 128 days of gestation, fetal sheep were exposed to Sham (n = 6) or I/R induced by cerebral ischemia for 30 min with reperfusion for 7 days (n = 5). Although I/R did not change the total number of IAIP+ cells in the cerebral cortex or white matter, cells with IAIP+ cytoplasm decreased, whereas cells with IAIP+ nuclei increased in the cortex. I/R reduced total neuronal number but did not change the IAIP+ neuronal number. The proportion of cytoplasmic IAIP+ neurons was reduced, but there was no change in the number of nuclear IAIP+ neurons. I/R increased the number of microglia and decreased the total numbers of IAIP+ microglia and nuclear IAIP+ microglia, but not the number of cytoplasmic IAIP+ microglia. I/R was associated with reduced numbers of oligodendrocytes and increased proliferating cells, without changes in the subcellular IAIP localization. IAIPs co-localized with the endoplasmic reticulum and histones. In conclusion, I/R alters the subcellular localization of IAIPs in cortical neurons and microglia but not in oligodendrocytes or proliferating cells. Taken together with the known neuroprotective effects of exogenous IAIPs, we speculate that endogenous IAIPs may play a role during recovery from I/R.

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.

Anti-Inflammatory Therapies for Treatment of Inflammation-Related Preterm Brain Injury.

Despite the prevalence of preterm brain injury, there are no established neuroprotective strategies to prevent or alleviate mild-to-moderate inflammation-related brain injury. Perinatal infection and inflammation have been shown to trigger acute neuroinflammation, including proinflammatory cytokine release and gliosis, which are associated with acute and chronic disturbances in brain cell survival and maturation. These findings suggest the hypothesis that the inhibition of peripheral immune responses following infection or nonspecific inflammation may be a therapeutic strategy to reduce the associated brain injury and neurobehavioral deficits. This review provides an overview of the neonatal immunity, neuroinflammation, and mechanisms of inflammation-related brain injury in preterm infants and explores the safety and efficacy of anti-inflammatory agents as potentially neurotherapeutics.

Non-additive effects of adjunct erythropoietin therapy with therapeutic hypothermia after global cerebral ischaemia in near-term fetal sheep.

KEY POINTS: Recombinant human erythropoietin (rEpo) is neuroprotective in immature animals, but it is unclear whether the combination of high-dose rEpo therapy with therapeutic hypothermia can further improve outcomes. Hypothermia and rEpo independently improved neuronal survival, with greater improvement with hypothermia, and similarly reduced numbers of caspase-3 positive cells and reactive microglia after 7 days recovery. Hypothermia, but not rEpo, was associated with markedly improved EEG power, whereas both interventions improved recovery of EEG frequency. There was no significant improvement in any outcome after combined rEpo and hypothermia compared with hypothermia alone, and of concern, the combination was associated with increased numbers of cortical caspase-3-positive cells compared with ischaemia-hypothermia. These data suggest that the mechanisms of neuroprotection with hypothermia and rEpo overlap and, thus, high-dose rEpo infusion does not appear to be an effective adjunct therapy for therapeutic hypothermia. ABSTRACT: Therapeutic hypothermia for hypoxic-ischaemic encephalopathy (HIE) provides incomplete neuroprotection. Recombinant human erythropoietin (rEpo) is neuroprotective in immature animals, but it is unclear whether adjunct rEpo therapy with therapeutic hypothermia can further improve outcomes. Near-term fetal sheep received sham-ischaemia (n = 9) or global cerebral ischaemia for 30 min (ischaemia-vehicle, n = 8), followed by intravenous infusion of rEpo (ischaemia-Epo, n = 8; 5000 U/kg loading dose, then 833.3 U/kg/h), cerebral hypothermia (ischaemia-hypothermia, n = 8), or rEpo plus hypothermia (ischaemia-Epo-hypothermia, n = 8), from 3 to 72 h post ischaemia. Fetal brains were collected 7 days after cerebral ischaemia. Cerebral ischaemia was associated with severe neuronal loss and microglial induction in the parasagittal cortex and subcortical regions. Hypothermia reduced overall neuronal loss, cortical caspase-3 and reactive microglia in the striatum and cortex, with greater recovery of electroencephalographic (EEG) power and spectral edge (SEF) from 48 h onwards. rEpo independently improved neuronal survival in the parasagittal cortex, hippocampal CA4 and thalamus, and reduced cortical caspase-3 and activated microglia in striatal and cortical areas, with greater SEF from 120 h onwards. However, ischaemia-Epo-hypothermia did not further improve outcomes compared with ischaemia-hypothermia and was associated with increased numbers of cortical caspase-3-positive cells. These findings suggest that although delayed, prolonged treatment with both hypothermia and rEpo are independently neuroprotective, they have overlapping anti-inflammatory and anti-apoptotic mechanisms, such that the delayed, high-dose rEpo infusion for 3 days did not materially augment neuroprotection with 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.

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.

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.

TLR7 agonist modulation of postasphyxial neurophysiological and cardiovascular adaptations in preterm fetal sheep.

Activation of Toll-like receptors (TLRs) after hypoxic-ischemic brain injury can exacerbate injury but also alleviate cell loss, as recently demonstrated with the TLR7 agonist Gardiquimod (GDQ). However, TLR agonists also modulate vascular function and neuronal excitability. Thus, we examined the effects of TLR7 activation with GDQ on cardiovascular function and seizures after asphyxia in preterm fetal sheep at 0.7 gestation (104 days, term ∼147 days). Fetuses received sham asphyxia or asphyxia induced by umbilical cord occlusion for 25 min or asphyxia followed by a continuous intracerebroventricular infusion of 3.34 mg of GDQ from 1 to 4 h after asphyxia. Fetuses were monitored continuously for 72 h postasphyxia. GDQ treatment was associated with sustained, moderate hypertension for 72 h (P < 0.05), with a transient increase in heart rate. Electroencephalographic (EEG) power was suppressed for the entire postasphyxial period in both groups, whereas EEG spectral edge transiently increased during the GDQ infusion compared with asphyxia alone (P < 0.05), with higher ß- and lower δ-EEG frequencies (P < 0.05). This increase in EEG frequency was not related to epileptiform activity. After the GDQ infusion, there was earlier onset of high-amplitude stereotypic evolving seizures, with increased numbers of seizures and seizure burden (P < 0.05). Hemodynamic function and seizure activity are important indices of preterm wellbeing. These data highlight the importance of physiological monitoring during preclinical testing of potential neuroprotective strategies.