Protection of axonal integrity with 48 or 72 h of cerebral hypothermia in near-term fetal sheep.

BACKGROUND: Therapeutic hypothermia is partially protective for neonatal hypoxic-ischemic encephalopathy (HIE). Damage to the white matter tracts is highly associated with adverse outcomes after HIE, but the effectiveness and optimal duration of hypothermia to attenuate axonal injury are unclear. METHODS: Near-term fetal sheep were randomized to sham control or cerebral ischemia for 30 min with normothermia or cerebral hypothermia from 3 to either 48 or 72 h. Sheep were killed after 7 days. SMI-312-labeled axons and myelin basic protein were quantified in the intragyral white matter of the first and second parasagittal gyri. RESULTS: Ischemia was associated with reduced axonal and myelin area fraction (p < 0.05); loss of axonal and myelin linearity (p < 0.05); and thin, sparse axons, with spheroids, compared to dense, linear morphology in sham controls and associated with induction of microglia in an amoeboid morphology. Both ischemia-48 h hypothermia and ischemia-72 h hypothermia improved axonal area fraction and linearity (p < 0.05), although abnormal morphological features were seen in a subset. Microglial induction was partially suppressed by ischemia-48 h hypothermia, with a ramified morphology. CONCLUSIONS: These data suggest that therapeutic hypothermia can alleviate post-ischemic axonopathy, in part by suppressing secondary inflammation.

Limited benefit of slow rewarming after cerebral hypothermia for global cerebral ischemia in near-term fetal sheep.

The optimal rate of rewarming after therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy is unknown, although it is widely suggested that slow rewarming is beneficial. Some preclinical studies suggest better outcomes with slower rewarming, but did not control for the duration of hypothermia. In this study, near-term fetal sheep (0.85 gestation) received 30 min cerebral ischemia followed by normothermia, 48 h hypothermia with rapid rewarming over 1 h, 48-h hypothermia with slow rewarming over 24 h, or 72-h hypothermia with rapid rewarming. Slow rewarming after 48 h of hypothermia improved recovery of EEG power compared with rapid rewarming (p < 0.05), but was not different from rapid rewarming after 72 h of hypothermia. At seven days recovery, neuronal survival was partially improved by both fast and slow rewarming after 48-h hypothermia, but less than 72-h hypothermia in the cortex and CA4 (p < 0.05). In conclusion, although electrographic recovery was partially improved by slow rewarming over 24 h following cerebral hypothermia for 48 h, optimal neuroprotection was seen with hypothermia for 72 h with rapid rewarming, suggesting that the overall duration of cooling was the critical determinant of outcomes after therapeutic hypothermia.

Time and sex dependent effects of magnesium sulphate on post-asphyxial seizures in preterm fetal sheep.

KEY POINTS: We evaluated the effect of magnesium sulphate (MgSO4 ) on seizures induced by asphyxia in preterm fetal sheep. MgSO4 did not prevent seizures, but significantly reduced the total duration, number of seizures, seizure amplitude and average seizure burden. Saline-asphyxia male fetuses had significantly more seizures than female fetuses, but male fetuses showed significantly greater reduction in seizures during MgSO4 infusion than female fetuses. A circadian profile of seizure activity was observed in all fetuses, with peak seizures seen around 04.00-06.00 h on the first and second days after the end of asphyxia. This study is the first to demonstrate that MgSO4 has utility as an anti-seizure agent after hypoxia-ischaemia. More information is needed about the mechanisms mediating the effect of MgSO4 on seizures and sexual dimorphism, and the influence of circadian rhythms on seizure expression. ABSTRACT: Seizures are common in newborns after asphyxia at birth and are often refractory to anti-seizure agents. Magnesium sulphate (MgSO4 ) has anticonvulsant effects and is increasingly given to women in preterm labour for potential neuroprotection. There is limited information on its effects on perinatal seizures. We examined the hypothesis that MgSO4 infusion would reduce fetal seizures after asphyxia in utero. Preterm fetal sheep at 0.7 gestation (104 days, term = 147 days) were given intravenous infusions of either saline (n = 14) or MgSO4 (n = 12, 160 mg bolus + 48 mg h-1 infusion over 48 h). Fetuses underwent umbilical cord occlusion (UCO) for 25 min, 24 h after the start of infusion. The start time for seizures did not differ between groups, but MgSO4 significantly reduced the total number of seizures (P < 0.001), peak seizure amplitude (P < 0.05) and seizure burden (P < 0.005). Within the saline-asphyxia group, male fetuses had significantly more seizures than females (P < 0.05). Within the MgSO4 -asphyxia group, although both sexes had fewer seizures than the saline-asphyxia group, the greatest effect of MgSO4 was on male fetuses, with reduced numbers of seizures (P < 0.001) and seizure burden (P < 0.005). Only 1 out of 6 MgSO4 males had seizures on the second day post-UCO compared to 5 out of 6 MgSO4 female fetuses (P = 0.08). Finally, seizures showed a circadian profile with peak seizures between 04.00 and 06.00 h on the first and second day post-UCO. Collectively, these results suggest that MgSO4 may have utility in treating perinatal seizures and has sexually dimorphic effects.

How long is sufficient for optimal neuroprotection with cerebral cooling after ischemia in fetal sheep?

The optimal duration of mild "therapeutic" hypothermia for neonates with hypoxic-ischemic encephalopathy is surprisingly unclear. This study assessed the relative efficacy of cooling for 48 h versus 72 h. Fetal sheep (0.85 gestation) received sham ischemia (n = 9) or 30 min global cerebral ischemia followed by normothermia (n = 8) or delayed hypothermia from 3 h to 48 h (n = 8) or 72 h (n = 8). Ischemia was associated with profound loss of electroencephalogram (EEG) power, neurons in the cortex and hippocampus, and oligodendrocytes and myelin basic protein expression in the white matter, with increased Iba-1-positive microglia and proliferation. Hypothermia for 48 h was associated with improved outcomes compared to normothermia, but a progressive deterioration of EEG power after rewarming compared to 72 h of hypothermia, with impaired neuronal survival and myelin basic protein, and more microglia in the white matter and cortex. These findings show that head cooling for 48 h is partially neuroprotective, but is inferior to cooling for 72 h after cerebral ischemia in fetal sheep. The close association between rewarming at 48 h, subsequent deterioration in EEG power and increased cortical inflammation strongly suggests that deleterious inflammation can be reactivated by premature rewarming.

Magnesium sulfate reduces EEG activity but is not neuroprotective after asphyxia in preterm fetal sheep.

Magnesium sulfate is now widely recommended for neuroprotection for preterm birth; however, this has been controversial because there is little evidence that magnesium sulfate is neuroprotective. Preterm fetal sheep (104 days gestation; term is 147 days) were randomly assigned to receive sham occlusion (n = 7), i.v. magnesium sulfate (n = 10) or saline (n = 8) starting 24 h before asphyxia until 24 h after asphyxia. Sheep were killed 72 h after asphyxia. Magnesium sulfate infusion reduced electroencephalograph power and fetal movements before asphyxia. Magnesium sulfate infusion did not affect electroencephalograph power during recovery, but was associated with marked reduction of the post-asphyxial seizure burden (mean ± SD: 34 ± 18 min vs. 107 ± 74 min, P < 0.05). Magnesium sulfate infusion did not affect subcortical neuronal loss. In the intragyral and periventricular white matter, magnesium sulfate was associated with reduced numbers of all (Olig-2+ve) oligodendrocytes in the intragyral (125 ± 23 vs. 163 ± 38 cells/field) and periventricular white matter (162 ± 39 vs. 209 ± 44 cells/field) compared to saline-treated controls ( P < 0.05), but no effect on microglial induction or astrogliosis. In conclusion, a clinically comparable dose of magnesium sulfate showed significant anticonvulsant effects after asphyxia in preterm fetal sheep, but did not reduce asphyxia-induced brain injury and exacerbated loss of oligodendrocytes.