Michael Reyes

Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic-ischemic encephalopathy.

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.

Optimizing Cerebral Autoregulation May Decrease Neonatal Regional Hypoxic-Ischemic Brain Injury.

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 (MAPOPT). Blood pressure deviation from MAPOPT 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 MAPOPT related to neurologic injury in several regions independent of birth asphyxia severity. Greater duration and deviation of blood pressure below MAPOPT were associated with greater injury in the paracentral gyri and white matter. Blood pressure within MAPOPT related to lesser injury in the white matter, putamen and globus pallidus, and brain stem. Finally, blood pressures that exceeded MAPOPT 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.

Impaired autophagosome clearance contributes to neuronal death in a piglet model of neonatal hypoxic-ischemic encephalopathy

To examine the temporal relationship of cortical autophagic flux with delayed neuronal cell death after hypoxia-ischemia (HI) in neonatal piglets. HI was produced with 45-min hypoxia and 7-min airway occlusion in 3–5-day-old piglets. Markers of autophagic, lysosomal and cell death signaling were studied via immunohistochemistry, immunoblotting, and histochemistry in piglet brains. In vitro, autophagy was impaired in cultured mouse cortical neurons treated with chloroquine with or without rapamycin for 1 d in the presence of Z-VAD-fmk, cyclosporine A, or vehicle control, and cell viability was assessed with the MTT assay. In vivo, neuronal cell death of sensorimotor cortex was delayed by 1–2 days after HI, whereas LC3-II, Beclin-1, PI3KC3, ATG12-ATG-5, and p-ULK1 increased by 1.5–6 h. Autophagosomes accumulated in cortical neurons by 1 d owing to enhanced autophagy and later to decreased autophagosome clearance, as indicated by LC3, Beclin-1, and p62 accumulation. Autophagy flux impairment was attributable to lysosomal dysfunction, as indicated by low lysosomal-associated membrane protein 2, cathepsin B, and cathepsin D levels at 1 d. Ubiquitin levels increased at 1 d. Autophagosome and p62 accumulated predominantly in neurons at 1 d, with p62 puncta occurring in affected cells. Beclin-1 colocalized with markers of caspase-dependent and caspase-independent apoptosis and necrosis in neurons. In vitro, mouse neonatal cortical neurons treated with rapamycin and chloroquine showed increased autophagosomes, but not autolysosomes, and increased cell death that was attenuated by cyclosporine A. Neonatal HI initially increases autophagy but later impairs autophagosome clearance, coinciding with delayed cortical neuronal death.

Hypothermia and Rewarming Activate a Macroglial Unfolded Protein Response Independent of Hypoxic-Ischemic Brain Injury in Neonatal Piglets.

Therapeutic hypothermia provides incomplete neuroprotection after hypoxia-ischemia (HI)-induced brain injury in neonates. We previously showed that cortical neuron and white matter apoptosis are promoted by hypothermia and early rewarming in a piglet model of HI. The unfolded protein response (UPR) may be one of the potential mediators of this cell death. Here, neonatal piglets underwent HI or sham surgery followed by 29 h of normothermia, 2 h of normothermia + 27 h of hypothermia or 18 h of hypothermia + rewarming. Piglets recovered for 29 h. Immunohistochemistry for endoplasmic reticulum to nucleus signaling-1 protein (ERN1), a marker of UPR activation, was used to determine the ratios of ERN1+ macroglia and neurons in the motor subcortical white matter and cerebral cortex. The ERN1+ macroglia were immunophenotyped as oligodendrocytes and astrocytes by immunofluorescent colabeling. Temperature (p = 0.046) and HI (p < 0.001) independently affected the ratio of ERN1+ macroglia. In sham piglets, sustained hypothermia (p = 0.011) and rewarming (p = 0.004) increased the ERN1+ macroglia ratio above that in normothermia. HI prior to hypothermia diminished the UPR. Ratios of ERN1+ macroglia correlated with white matter apoptotic profile counts in shams (r = 0.472; p = 0.026), thereby associating UPR activation with white matter apoptosis during hypothermia and rewarming. Accordingly, macroglial cell counts decreased in shams that received sustained hypothermia (p = 0.009) or rewarming (p = 0.007) compared to those in normothermic shams. HI prior to hypothermia neutralized the macroglial cell loss. Neither HI nor temperature affected ERN1+ neuron ratios. In summary, delayed hypothermia and rewarming activate the macroglial UPR, which is associated with white matter apoptosis. HI may decrease the macroglial endoplasmic reticulum stress response after hypothermia and rewarming.

Sex-specific associations between cerebrovascular blood pressure autoregulation and cardiopulmonary injury in neonatal encephalopathy and therapeutic hypothermia.

Background:Cardiopulmonary injury is common in neonatal encephalopathy, but the link with cerebrovascular dysfunction is unknown. We hypothesized that alterations of cerebral autoregulation are associated with cardiopulmonary injury in neonates treated with therapeutic hypothermia (TH) for neonatal encephalopathy.Methods:The cerebral hemoglobin volume index (HVx) from near-infrared spectroscopy was used to identify the mean arterial blood pressure (MAP) with optimal autoregulatory vasoreactivity (MAPOPT). We measured associations between MAP relative to MAPOPT and indicators of cardiopulmonary injury (duration of mechanical respiratory support and administration of inhaled nitric oxide (iNO), milrinone, or steroids).Results:We identified associations between cerebrovascular autoregulation and cardiopulmonary injury that were often sex-specific. Greater MAP deviation above MAPOPT was associated with shorter duration of intubation in boys but longer ventilatory support in girls. Greater MAP deviation below MAPOPT related to longer intensive care stay in boys. Milrinone was associated with greater MAP deviation below MAPOPT in girls.Conclusion:MAP deviation from MAPOPT may relate to cardiopulmonary injury after neonatal encephalopathy, and sex may modulate this relationship. Whereas MAP above MAPOPT may protect the brain and lungs in boys, it may be related to cardiopulmonary injury in girls. Future studies are needed to characterize the role of sex in these associations.

Relationships between cerebral autoregulation and markers of kidney and liver injury in neonatal encephalopathy and therapeutic hypothermia

Objective:We studied whether cerebral blood pressure autoregulation and kidney and liver injuries are associated in neonatal encephalopathy (NE).Study design:We monitored autoregulation of 75 newborns who received hypothermia for NE in the neonatal intensive care unit to identify the mean arterial blood pressure with optimized autoregulation (MAPOPT). Autoregulation parameters and creatinine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed using adjusted regression models.Results:Greater time with blood pressure within MAPOPT during hypothermia was associated with lower creatinine in girls. Blood pressure below MAPOPT related to higher ALT and AST during normothermia in all neonates and boys. The opposite occurred in rewarming when more time with blood pressure above MAPOPT related to higher AST.Conclusions:Blood pressures that optimize cerebral autoregulation may support the kidneys. Blood pressures below MAPOPT and liver injury during normothermia are associated. The relationship between MAPOPT and AST during rewarming requires further study.

Abdominal near-infrared spectroscopy in a piglet model of gastrointestinal hypoxia produced by graded hypoxia or superior mesenteric artery ligation

BackgroundAbdominal near-infrared spectroscopy (aNIRS) may detect gastrointestinal hypoxia before necrotizing enterocolitis develops. We sought to validate aNIRS during splanchnic hypoxia and hypoperfusion in neonatal piglets.MethodsAnesthetized piglets underwent systemic hypoxia or 3 h superior mesenteric artery (SMA) ligation with aNIRS monitoring.ResultsDuring progressive hypoxia, gastrointestinal tissue oxyhemoglobin saturation measured by aNIRS decreased linearly with oxyhemoglobin saturation measured directly in the portal vein. Correlation coefficients were 0.94–0.99 in each of 10 piglets, the average regression slope of 0.73 (95% confidence interval: 0.57, 0.89) differed from one (P<0.004), and the intercept on the aNIRS axis of 9.5% (4.4, 14.6) differed from zero (P<0.0025). Umbilical venous oxyhemoglobin saturation also correlated strongly with the portal vein oxyhemoglobin saturation (r=0.83–0.99), with a slope not different from one. SMA ligation caused ileal blood flow to decrease by ~50%, and produced a sustained decrease in aNIRS oximetry from approximately 60 to 30%.ConclusionaNIRS can detect abrupt and sustained gastrointestinal hypoperfusion associated with arterial occlusion in a neonatal model. The highly linear relationship of portal venous oxyhemoglobin saturation with aNIRS and umbilical vein saturation during graded hypoxia implies that these measures can accurately track tissue oxygenation trends over a wide range in individual subjects.

Cerebrovascular blood pressure autoregulation monitoring and postoperative transient ischemic attack in pediatric moyamoya vasculopathy

Children with moyamoya vasculopathy are at high risk of perioperative cerebral ischemia or hyperperfusion. Maintaining blood pressure within the range of functional cerebrovascular blood pressure autoregulation might reduce the risk of perioperative neurologic injury.