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.

Asymmetrical nasal high flow ventilation improves clearance of CO2 from the anatomical dead space and increases positive airway pressure.

Positive airway pressure that dynamically changes with breathing, and clearance of anatomical dead space are the key mechanisms of noninvasive respiratory support with nasal high flow (NHF). Pressure mainly depends on flow rate and nare occlusion. The hypothesis is that an increase in asymmetrical occlusion of the nares leads to an improvement in dead-space clearance resulting in a reduction in re-breathing. Clearance was investigated with volumetric capnography in an adult upper-airway model, which was ventilated by a lung simulator with entrained carbon dioxide (CO2) at respiratory rates (RR) of 15-45 min-1 and at 18 min-1 with chronic obstructive pulmonary disease (COPD) breathing patterns. Clearance was assessed at NHF of 20-60 L/min with a symmetrical interface (SI) and an asymmetrical interface (AI). CO2 kinetics visualized by infrared spectroscopy and mathematical modeling were used to study the mechanisms of clearance. At a higher RR (35 min-1) and NHF of 60 L/min, clearance in the upper airway was significantly higher with the AI when compared with the SI (29.64 ± 9.96%, P < 0.001), as opposed to at a lower RR (15 min-1) (1.40 ± 6.25%, P > 0.05), (means ± SD). With COPD breathing, clearance by NHF was reduced but significantly improved with the AI by 45.93% relative to the SI at NHF 20 L/min (P < 0.0001). The maximum pressure achieved with the AI was 6.6 cmH2O and NHF was 60 L/min at the end of expiration. Pressure differences between nasal cavities led to the reverse flow observed in the optical model. Asymmetrical NHF increases dead-space clearance by reverse flow through the choanae and accelerates purging of expired gas via the less occluded nare.NEW & NOTEWORTHY The asymmetrical interface generated reverse flow in the nasal cavities and across the choana, which led to unidirectional purging of expired gas from the upper airways. This accelerated the clearance of anatomical dead space and reduced re-breathing while increased resistance to flow resulted in higher positive end-expiratory pressure (PEEP). These findings are relevant to patients with elevated respiratory rates or with expiratory flow limitations where dead-space clearance by NHF can be substantially reduced.

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.

Window of opportunity for human amnion epithelial stem cells to attenuate astrogliosis after umbilical cord occlusion in preterm fetal sheep.

There is increasing evidence that administration of many types of stem cells, including human amnion epithelial cells (hAECs), can reduce hypoxic-ischemic injury, including in the perinatal brain. However, the therapeutic window for single dose treatment is not known. We compared the effects of early and delayed intracerebroventricular administration of hAECs in fetal sheep at 0.7 gestation on brain injury induced by 25 minutes of complete umbilical cord occlusion (UCO) or sham occlusion. Fetuses received either 1 × 106 hAECs or vehicle alone, as an infusion over 1 hour, either 2 or 24 hours after UCO. Fetuses were killed for brain histology at 7 days post-UCO. hAEC infusion at both 2 and 24 hours had dramatic anti-inflammatory and anti-gliotic effects, including significantly attenuating the increase in microglia after UCO in the white and gray matter and the number of astrocytes in the white matter. Both protocols partially improved myelination, but had no effect on total or immature/mature numbers of oligodendrocytes. Neuronal survival in the hippocampus was increased by hAEC infusion at either 2 or 24 hours, whereas only hAECs at 24 hours were associated with improved neuronal survival in the striatum and thalamus. Neither protocol improved recovery of electroencephalographic (EEG) power. These data suggest that a single infusion of hAECs is anti-inflammatory, anti-gliotic, and neuroprotective in preterm fetal sheep when given up to 24 hours after hypoxia-ischemia, but was associated with limited white matter protection after 7 days recovery and no improvement in the recovery of EEG power.

Protective effects of delayed intraventricular TLR7 agonist administration on cerebral white and gray matter following asphyxia in the preterm fetal sheep.

Preterm brain injury is highly associated with inflammation, which is likely related in part to sterile responses to hypoxia-ischemia. We have recently shown that neuroprotection with inflammatory pre-conditioning in the immature brain is associated with induction of toll-like receptor 7 (TLR7). We therefore tested the hypothesis that central administration of a synthetic TLR7 agonist, gardiquimod (GDQ), after severe hypoxia-ischemia in preterm-equivalent fetal sheep would improve white and gray matter recovery. Fetal sheep at 0.7 of gestation received sham asphyxia or asphyxia induced by umbilical cord occlusion for 25 minutes, followed by a continuous intracerebroventricular infusion of GDQ or vehicle from 1 to 4 hours (total dose 1.8 mg/kg). Sheep were killed 72 hours after asphyxia for histology. GDQ significantly improved survival of immature and mature oligodendrocytes (2',3'-cyclic-nucleotide 3'-phosphodiesterase, CNPase) and total oligodendrocytes (oligodendrocyte transcription factor 2, Olig-2) within the periventricular and intragyral white matter. There were reduced numbers of cells showing cleaved caspase-3 positive apoptosis and astrogliosis (glial fibrillary acidic protein, GFAP) in both white matter regions. Neuronal survival was increased in the dentate gyrus, caudate and medial thalamic nucleus. Central infusion of GDQ was associated with a robust increase in fetal plasma concentrations of the anti-inflammatory cytokines, interferon-ß (IFN-ß) and interleukin-10 (IL-10), with no significant change in the concentration of the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α). In conclusion, delayed administration of the TLR7 agonist, GDQ, after severe hypoxia-ischemia in the developing brain markedly ameliorated white and gray matter damage, in association with upregulation of anti-inflammatory cytokines. These data strongly support the hypothesis that modulation of secondary inflammation may be a viable therapeutic target for injury of the preterm brain.

Delayed intranasal infusion of human amnion epithelial cells improves white matter maturation after asphyxia in preterm fetal sheep.

Perinatal hypoxic-ischemic (HI) brain injury remains highly associated with neurodevelopmental disability after preterm birth. There is increasing evidence that disability is linked with impaired white matter maturation, but there is no specific treatment. In this study, we evaluated whether, in preterm fetal sheep, delayed intranasal infusion of human amnion epithelial cells (hAECs) given 1, 3 and 10 days after severe HI, induced by umbilical cord occlusion for 25 min, can restore white matter maturation or reduce delayed cell loss. After 21 days recovery, asphyxia was associated with reduced electroencephalographic (EEG) maturation, brain weight and cortical area, impaired maturation of oligodendrocytes (OLs), no significant loss of total OLs but a marked reduction in immature/mature OLs and reduced myelination. Intranasal infusion of hAECs was associated with improved brain weight and restoration of immature/mature OLs and fractional area of myelin basic protein, with reduced microglia and astrogliosis. Cortical EEG frequency distribution was partially improved, with reduced loss of cortical area, and attenuated cleaved-caspase-3 expression and microgliosis. Neuronal survival in deep grey matter nuclei was improved, with reduced microglia, astrogliosis and cleaved-caspase-3-positive apoptosis. These findings suggest that delayed intranasal hAEC administration has potential to alleviate chronic dysmaturation after perinatal HI.

Magnesium sulphate and cardiovascular and cerebrovascular adaptations to asphyxia in preterm fetal sheep.

Magnesium sulphate is a standard therapy for eclampsia in pregnancy and is widely recommended for perinatal neuroprotection during threatened preterm labour. MgSO4 is a vasodilator and negative inotrope. Therefore the aim of this study was to investigate the effect of MgSO4 on the cardiovascular and cerebrovascular responses of the preterm fetus to asphyxia. Fetal sheep were instrumented at 98 ± 1 days of gestation (term = 147 days). At 104 days, unanaesthetised fetuses were randomly assigned to receive an intravenous infusion of MgSO4 (n = 6) or saline (n = 9). At 105 days all fetuses underwent umbilical cord occlusion for 25 min. Before occlusion, MgSO4 treatment reduced heart rate and increased femoral blood flow (FBF) and vascular conductance compared to controls. During occlusion, carotid and femoral arterial conductance and blood flows were higher in MgSO4-treated fetuses than controls. After occlusion, fetal heart rate was lower and carotid and femoral arterial conductance and blood flows were higher in MgSO4-treated fetuses than controls. Femoral arterial waveform height and width were increased during MgSO4 infusion, consistent with increased stroke volume. MgSO4 did not alter the fetal neurophysiological or nuchal electromyographic responses to asphyxia. These data demonstrate that a clinically comparable dose of MgSO4 increased FBF and stroke volume without impairing mean arterial pressure (MAP) or carotid blood flow (CaBF) during and immediately after profound asphyxia. Thus, MgSO4 may increase perfusion of peripheral vascular beds during adverse perinatal events.

Therapeutic Hypothermia for Neonatal Hypoxic-Ischemic Encephalopathy - Where to from Here?

Hypoxia-ischemia before or around the time of birth occurs in approximately 2/1000 live births and is associated with a high risk of death or lifelong disability. Therapeutic hypothermia is now well established as standard treatment for infants with moderate to severe hypoxic-ischemic encephalopathy but is only partially effective. There is compelling preclinical and clinical evidence that hypothermia is most protective when it is started as early as possible after hypoxia-ischemia. Further improvements in outcome from therapeutic hypothermia are very likely to arise from strategies to reduce the delay before starting treatment of affected infants. In this review, we examine evidence that current protocols are reasonably close to the optimal depth and duration of cooling, but that the optimal rate of rewarming after hypothermia is unclear. The potential for combination treatments to augment hypothermic neuroprotection has considerable promise, particularly with endogenous targets such as melatonin and erythropoietin, and noble gases such as xenon. We dissect the critical importance of preclinical studies using realistic delays in treatment and clinically relevant cooling protocols when examining combination treatment, and that for many strategies overlapping mechanisms of action can substantially attenuate any effects.

Synergistic white matter protection with acute-on-chronic endotoxin and subsequent asphyxia in preterm fetal sheep.

BACKGROUND: Perinatal asphyxia and exposure to intrauterine infection are associated with impaired neurodevelopment in preterm infants. Acute exposure to non-injurious infection and/or inflammation can either protect or sensitize the brain to subsequent hypoxia-ischemia. However, the effects of subacute infection and/or inflammation are unclear. In this study we tested the hypothesis that acute-on-chronic exposure to lipopolysaccharide (LPS) would exacerbate white matter injury after subsequent asphyxia in preterm fetal sheep. METHODS: Fetal sheep at 0.7 gestational age received a continuous LPS infusion at 100 ng/kg for 24 hours, then 250 ng/kg/24 hours for 96 hours, plus 1 µg boluses of LPS at 48, 72, and 96 hours or the same volume of saline. Four hours after the last bolus, complete umbilical cord occlusion or sham occlusion was induced for 15 minutes. Sheep were sacrificed 10 days after the start of infusions. RESULTS: LPS exposure was associated with induction of microglia and astrocytes and loss of total and immature and mature oligodendrocytes (n = 9) compared to sham controls (n = 9). Umbilical cord occlusion with saline infusions was associated with induction of microglia, astrogliosis, and loss of immature and mature oligodendrocytes (n = 9). LPS exposure before asphyxia (n = 8) was associated with significantly reduced microglial activation and astrogliosis and improved numbers of immature and mature oligodendrocytes compared to either LPS exposure or asphyxia alone. CONCLUSIONS: Contrary to our initial hypothesis, the combination of acute-on-chronic LPS with subsequent asphyxia reduced neuroinflammation and white matter injury compared with either intervention alone.

Status epilepticus after prolonged umbilical cord occlusion is associated with greater neural injury in [corrected] fetal sheep at term-equivalent.

The majority of pre-clinical studies of hypoxic-ischemic encephalopathy at term-equivalent have focused on either relatively mild insults, or on functional paradigms of cerebral ischemia or hypoxia-ischemia/hypotension. There is surprisingly little information on the responses to single, severe 'physiological' insults. In this study we examined the evolution and pattern of neural injury after prolonged umbilical cord occlusion (UCO). 36 chronically instrumented fetal sheep at 125-129 days gestational age (term = 147 days) were subjected to either UCO until mean arterial pressure was < = 8 mmHg (n = 29), or sham occlusion (n = 7). Surviving fetuses were killed after 72 hours for histopathologic assessment with acid-fuchsin thionine. After UCO, 11 fetuses died with intractable hypotension and 5 ewes entered labor and were euthanized. The remaining 13 fetuses showed marked EEG suppression followed by evolving seizures starting at 5.8 (6.8) hours (median (interquartile range)). 6 of 13 developed status epilepticus, which was associated with a transient secondary increase in cortical impedance (a measure of cytotoxic edema, p<0.05). All fetuses showed moderate to severe neuronal loss in the hippocampus and the basal ganglia but mild cortical cell loss (p<0.05 vs sham occlusion). Status epilepticus was associated with more severe terminal hypotension (p<0.05) and subsequently, greater neuronal loss (p<0.05). In conclusion, profound UCO in term-equivalent fetal sheep was associated with delayed seizures, secondary cytotoxic edema, and subcortical injury, consistent with the predominant pattern after peripartum sentinel events at term. It is unclear whether status epilepticus exacerbated cortical injury or was simply a reflection of a longer duration of asphyxia.

nNOS inhibition during profound asphyxia reduces seizure burden and improves survival of striatal phenotypic neurons in preterm fetal sheep.

Basal ganglia injury after hypoxia-ischemia remains common in preterm infants, and is closely associated with later cerebral palsy. In the present study we tested the hypothesis that a highly selective neuronal nitric oxide synthase (nNOS) inhibitor, JI-10, would improve survival of striatal phenotypic neurons after profound asphyxia, and that the subsequent seizure burden and recovery of EEG are associated with neural outcome. 24 chronically instrumented preterm fetal sheep were randomized to either JI-10 (3 ml of 0.022 mg/ml, n = 8) or saline (n = 8) infusion 15 min before 25 min complete umbilical cord occlusion, or saline plus sham-occlusion (n = 8). Umbilical cord occlusion was associated with reduced numbers of calbindin-28k-, GAD-, NPY-, PV-, Calretinin- and nNOS-positive striatal neurons (p < 0.05 vs. sham occlusion) but not ChAT-positive neurons. JI-10 was associated with increased numbers of calbindin-28k-, GAD-, nNOS-, NPY-, PV-, Calretinin- and ChAT-positive striatal neurons (p < 0.05 vs. saline + occlusion). Seizure burden was strongly associated with loss of calbindin-positive cells (p < 0.05), greater seizure amplitude was associated with loss of GAD-positive cells (p < 0.05), and with more activated microglia in the white matter tracts (p < 0.05). There was no relationship between EEG power after 7 days recovery and total striatal cell loss, but better survival of NPY-positive neurons was associated with lower EEG power. In summary, these findings suggest that selective nNOS inhibition during asphyxia is associated with protection of phenotypic striatal projection neurons and has potential to help reduce basal ganglia injury in some premature babies.

Partial neural protection with prophylactic low-dose melatonin after asphyxia in preterm fetal sheep.

Melatonin is a naturally occurring indolamine with mild antioxidant properties that is neuroprotective in perinatal animals. There is limited information on its effects on preterm brain injury. In this study, 23 chronically instrumented fetal sheep received 25 minutes of complete umbilical cord occlusion at 101 to 104 days gestation (term is 147 days). Melatonin was administered to the ewe 15 minutes before occlusion (0.1 mg/kg bolus followed by 0.1 mg/kg per hour for 6 hours, n=8), or the equivalent volume of vehicle (2% ethanol, n=7), or saline (n=8), or maternal saline plus sham occlusion (n=8). Sheep were killed after 7 days recovery in utero. Fetal blood pressure, heart rate, nuchal activity, and temperature were similar between groups. Vehicle infusion was associated with improved neuronal survival in the caudate nucleus, but greater neuronal loss in the regions of the hippocampus, with reduced proliferation and increased ameboid microglia in the white matter (P<0.05). Maternal melatonin infusion was associated with faster recovery of fetal EEG, prolonged reduction in carotid blood flow, similar neuronal survival to vehicle, improved numbers of mature oligodendrocytes, and reduced microglial activation in the white matter (P<0.05). Prophylactic maternal melatonin treatment is partially protective but its effects may be partly confounded by ethanol used to dissolve melatonin.

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

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