The results of the present study failed to support our hypothesis that early treatment with the antioxidant EUK-134 at 30 minutes after reoxygenation from neonatal HI would have an additive neuroprotective benefit with hypothermia that was delayed by a clinically relevant duration of 4 hours. The endpoint of viable neurons was the measure of neuroprotection and was assessed using two different cell counting methods. Both methods yielded similar results. Thus, despite the limitations of profile counting-based methods, this approach is nevertheless valid. Several possibilities need to be discussed to interpret this inefficacy of EUK-134 to augment hypothermic neuroprotection.
The subpopulation of striatal neurons protected by EUK-134 may be the same subpopulation that is able to be protected by delayed hypothermia. Thus, a different subpopulation of striatal neurons that are highly vulnerable to HI might not be protected by either intervention. The operative mechanisms for hypothermic neuroprotection could be numerous, and the exact mechanisms have not been identified clearly. Because there was no detectable additive effect, our results suggest that EUK-134 and hypothermia may have a common mechanism of blocking oxidative stress, either by attenuating superoxide/hydrogen peroxide production or by increasing scavenging of these oxidants.
Another possibility is that the level of oxidative stress in a subpopulation of neurons may already induce necrosis or downstream cell death signaling that can no longer be reversed when treatment with EUK-134 is delayed by 30 minutes. We did not administer EUK-134 immediately after reoxygenation because such a short delay would not be clinically feasible. Moreover, administration of EUK-134 at 30 minutes was effective in reducing protein nitration and carbonyl formation that are normally seen at 3 hours after HI. Nevertheless, neuronal nitric oxide synthase is already increased in the membrane fraction of striatal homogenates by 5 minutes after reoxygenation23
and may already be producing substantial nitrative and oxidative stress by 30 minutes in a subpopulation of neurons. In addition, phosphorylation of NR1 of the N-Methyl-d-Aspartate receptor is already evident by 5 minutes after reoxygenation23
and may lead to augmented calcium entry. Phosphorylation of NR1 at Ser897 may be important, because it has been reported that a D1 receptor antagonist28
or induction of hypothermia at 5 minutes of recovery23
reduces HI-induced phosphorylation at this protein kinase A-sensitive site and is associated with protection of piglet striatal neurons. Thus, an antioxidant may need to be administered immediately upon reoxygenation to obtain maximum efficacy and permit an additive neuroprotective effect with delayed hypothermia. Furthermore, EUK-134 is a manganese-salen–based antioxidant that was selected because of its efficacy in an adult rat stroke model30
and its ability to reduce markers of oxidative stress in the current piglet model.24
Nevertheless, newer generations of manganese-based catalytic antioxidants27,31
might be more effective than EUK-134 and provide additive protection with hypothermia. Alternatively, the efficacy of EUK-134 may have been greater if the piglets had been maintained on 100% O2
during early recovery, which is often practiced clinically and which may augment oxidative stress. In the present study, 50% O2
was used for resuscitation to offset the acidosis-induced shift in the oxyhemoglobin dissociation curve, and the inspired O2
concentration was gradually reduced to 30% as the acidosis was corrected over the first 30 minutes of ROSC.
Another consideration is that hypothermia is thought to protect the brain by multiple mechanisms,32
and demonstration of statistical significance of additional protection by another therapy may be difficult. Interestingly, ventilation with 50% xenon immediately during recovery has been shown to exert a statistically significant additive neuroprotective effect with immediate induction of hypothermia after HI in piglets.9
Thus, there may be mechanisms of injury that are not targeted by hypothermia but that are targeted by another therapy such as xenon. However, in a model of HI induced by 30-minute hypoxia followed by 7-minute asphyxia, immediate induction of hypothermia was able to completely block loss of viable neurons in putamen.7
Although this duration of hypoxia was less severe than the 40-minute duration that we used, these previous results indicate that hypothermia can target the multiple mechanisms involved in piglet striatum injury when it is initiated rapidly.
Another point to consider is that the duration of hypothermia was limited to 1 day because of the logistical difficulty of maintaining paralyzed, sedated piglets on a ventilator for multiple nights in a laboratory intensive care setting. In the clinical setting, therapeutic hypothermia is usually maintained for 2–3 days. When the onset of hypothermia is delayed and the evolution of the injury process has already progressed, a longer duration of hypothermia may be required to achieve maximum benefits in preventing delayed neurodegeneration.32
Finally, it is conceivable that hypothermia or prolonged sedation with fentanyl has adverse side effects that prevent additive neuroprotection with EUK-134. However, in sham-operated piglets treated with saline, EUK-134, hypothermia, or EUK-134 plus hypothermia, we did not see evidence of abnormal histopathology. This finding is consistent with previous work in which normothermic and hypothermic piglets were sedated with fentanyl for 24 hours.7
Moreover, a positive effect of delayed hypothermia was able to be detected when both normothermic- and hypothermic-treated encephalopathic newborns received sedation with opioids.4
With a 4-hour delay in instituting hypothermia, significant but incomplete neuroprotection was evident in striatum in the absence of EUK-134. This result is consistent with improved outcome in clinical studies in which the delay to induction of hypothermia after birth was usually 3–6 hours.1,2,4
However in a different piglet model of HI involving 45 minutes of more severe hypoxia (5–6% inspired oxygen), delaying hypothermia by 3 hours after reoxygenation was no longer significantly effective in improving global histopathological scores.13
One possible explanation is that the therapeutic time window for hypothermia is shorter when the insult is more severe. This severity-related impotency of hypothermia would be consistent with the clinical head cooling trial, which found that hypothermia instituted within 6 hours of birth was less effective in those presenting with more severe symptoms.2
Furthermore, analysis of magnetic resonance images indicated that hypothermia reduced the incidence and severity of basal ganglia and thalamic lesions to a greater extent in newborns with moderate reductions in amplitude-integrated electroencephalographic activity than in those presenting with severe reductions during the first 6 hours after birth.17
In contrast, a recent trial with robust whole-body cooling was able to demonstrate efficacy in those with severe encephalopathy despite an average delay in initiating hypothermia of 5 hours after birth and an additional 1.6 hours to achieve the target temperature.4
In previous work with the same model of HI in piglets, administration of EUK-134 at 30 minutes after reoxygenation increased the number of viable neurons in putamen at 4 days of recovery to 41% of that in sham-operated piglets.24
In the present study, the recovery period was extended to 10 days. Treatment with the same dose of EUK-134 at 30 minutes after reoxygenation resulted in 40% of the neurons remaining viable. Consistent with previous work,19,20
neuronal loss was more severe in putamen than in caudate nucleus in the normothermic HI group. Despite the less severe neuronal loss, statistically significant increases in the number of viable neurons still could be detected with EUK-134, hypothermia, and combined treatment in caudate nucleus. Therefore, the benefit of these treatments occurred throughout the striatum. The magnitude of the improvement with EUK-134 treatment after 10 days of recovery is similar to what was previously found in caudate nucleus after a 4-day recovery period.24
Thus, the protection afforded by EUK-134 and hypothermia appear to be sustained.
The peri-Rolandic neocortex is also selectively vulnerable to HI in term newborns; this vulnerability has been attributed to developmentally regulated increases in metabolic energy demand.33
In piglets, the parasagittal neocortex at the coronal level of the striatum has selectively high activity of cytochrome oxidase.19
This observation is consistent with metabolic development of primary sensorimotor cortex preceding metabolic development in other areas of neocortex at this stage of development in piglets. This area of neocortex is also selectively vulnerable in piglets, and this selectivity is likely related to high metabolic activity in this region rather than a vascular watershed distribution. In the present study, neuronal viability 10 days after HI in the normothermic group was significantly reduced to about half of that in the sham animals, whereas values in the three HI posttreatment groups were not significantly different from the value in the sham group. Although this result suggests that each of the treatments benefited this vulnerable region of neocortex, the values in the three HI posttreatment groups were not significantly different from that in the normothermic HI group, in part, because of the high variability of injury in this region with the present model. This variability may be associated with the presence of electrical seizure activity. Our study cannot address this because electroencephalographic activity was not monitored. Moreover, unbiased stereology was not performed in cortex to correct for possible expansion or contraction of injured tissue. However, in this model of selective neuronal vulnerability, expansion or shrinkage of brain structures is not prominent.
Many piglets had detectable neurological deficits at 4 days of recovery from HI. The median values of the three treatment groups were similar and were below the 25th percentile of the normothermia-saline group. However, the values were not significantly different because of the considerable variability among animals, and a larger sample size may be needed to demonstrate significant differences.
Hypoxia and asphyxia produced variable increases in blood glucose concentration that may have contributed to the variability in outcome. However, we previously found that hyperglycemia was not associated with adverse outcome; rather, decreases in glucose below 80 mg/dL after resuscitation were associated with worse neurological deficits.34
Therefore, piglets received 5% dextrose infusion to maintain glucose stores and prevent hypoglycemia. Moreover, hyperglycemia has not been found to adversely affect outcome in other models of ischemic injury in immature brain as it does in mature brain.35,36
In summary, treatment of newborn piglets with the antioxidant EUK-134 at 30 minutes after reoxygenation or treatment with hypothermia at 34°C for one day starting at 4 hours of recovery from HI partially protected striatal neurons. However, combining early antioxidant treatment with delayed hypothermia did not produce a statistically superior benefit, and the confidence intervals suggest that any true additional benefit would unlikely be meaningful. Other approaches will be necessary to extend the therapeutic window for induced hypothermia in newborns after HI.