The consequences of therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy are poorly understood. Adverse effects from suboptimal rewarming could diminish neuroprotection from hypothermia. Therefore, we tested whether rewarming is associated with apoptosis. Piglets underwent hypoxia-asphyxia followed by normothermic or hypothermic recovery at 2 hours. Hypothermic groups were divided into those with no rewarming, rewarming at 0.5 °C/hour, or rewarming at 4 °C/hour. Neurodegeneration at 29 hours was assessed by hematoxylin and eosin staining, TUNEL assay, and immunoblotting for cleaved caspase-3. Rewarmed piglets had more apoptosis in motor cortex than did those that remained hypothermic after hypoxia-asphyxia. Apoptosis in piriform cortex was greater in hypoxic-asphyxic, rewarmed piglets than in naive/sham piglets. Caspase-3 inhibitor suppressed apoptosis with rewarming. Rapidly rewarmed piglets had more caspase-3 cleavage in cerebral cortex than did piglets that remained hypothermic or piglets that were rewarmed slowly. We conclude that rewarming from therapeutic hypothermia can adversely affect the newborn brain by inducing apoptosis through caspase mechanisms.

Background: Therapeutic hypothermia provides incomplete neuroprotection for neonatal hypoxic-ischemic encephalopathy (HIE). We examined whether hemodynamic goals that support autoregulation are associated with decreased brain injury and whether these relationships are affected by birth asphyxia or vary by anatomic region. Methods: Neonates cooled for HIE received near-infrared spectroscopy autoregulation monitoring to identify the mean arterial blood pressure with optimized autoregulatory function (MAP_OPT). Blood pressure deviation from MAP_OPT was correlated with brain injury on MRI after adjusting for the effects of arterial carbon dioxide, vasopressors, seizures, and birth asphyxia severity. Results: Blood pressure deviation from MAP_OPT related to neurologic injury in several regions independent of birth asphyxia severity. Greater duration and deviation of blood pressure below MAP_OPT were associated with greater injury in the paracentral gyri and white matter. Blood pressure within MAP_OPT related to lesser injury in the white matter, putamen and globus pallidus, and brain stem. Finally, blood pressures that exceeded MAP_OPT were associated with reduced injury in the paracentral gyri. Conclusions: Blood pressure deviation from optimal autoregulatory vasoreactivity was associated with MRI markers of brain injury that, in many regions, were independent of the initial birth asphyxia. Targeting hemodynamic ranges to optimize autoregulation has potential as an adjunctive therapy to hypothermia for HIE.

Therapeutic hypothermia is widely used to treat neonatal hypoxic ischemic (HI) brain injuries. However, potentially deleterious effects of delaying the induction of hypothermia and of rewarming on white matter injury remain unclear. We used a piglet model of HI to assess the effects of delayed hypothermia and rewarming on white matter apoptosis. Piglets underwent HI injury or sham surgery followed by normothermic or hypothermic recovery at 2 h. Hypothermic groups were divided into those with no rewarming, slow rewarming at 0.5°C/h, or rapid rewarming at 4°C/h. Apoptotic cells in the subcortical white matter of the motor gyrus, corpus callosum, lateral olfactory tract, and internal capsule at 29 h were identified morphologically and counted by hematoxylin & eosin staining. Cell death was verified by TUNEL assay. White matter neurons were also counted, and apoptotic cells were immunophenotyped with the oligodendrocyte marker 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase). Hypothermia, slow rewarming, and rapid rewarming increased apoptosis in the subcortical white matter relative to normothermia (p<0.05). The number of white matter neurons was not lower in groups with more apoptosis after hypothermia or rapid rewarming, indicating that the apoptosis occurred among glial cells. Hypothermic piglets had more apoptosis in the lateral olfactory tract than those that were rewarmed (p<0.05). The promotion of apoptosis by hypothermia and rewarming in these regions was independent of HI. In the corpus callosum, HI piglets had more apoptosis than shams after normothermia, slow rewarming, and rapid rewarming (p<0.05). Many apoptotic cells were myelinating oligodendrocytes identified by CNPase positivity. Our results indicate that delaying the induction of hypothermia and rewarming are associated with white matter apoptosis in a piglet model of HI; in some regions these temperature effects are independent of HI. Vulnerable cells include myelinating oligodendrocytes. This study identifies a deleterious effect of therapeutic hypothermia in the developing brain.