Focal Brain Injury Associated with a Model of Severe Hypoxic-Ischemic Encephalopathy in Nonhuman Primates

Abstract

Worldwide, hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal mortality and morbidity. To better understand the mechanisms contributing to brain injury and improve outcomes in neonates with HIE, better preclinical animal models that mimic the clinical situation following birth asphyxia in term newborns are needed. In an effort to achieve this goal, we modified our nonhuman primate model of HIE induced by in utero umbilical cord occlusion (UCO) to include postnatal hypoxic episodes, in order to simulate apneic events in human neonates with HIE. We describe a cohort of 4 near-term fetal Macaca nemestrina that underwent 18 min of in utero UCO, followed by cesarean section delivery, resuscitation, and subsequent postnatal mechanical ventilation, with exposure to intermittent daily hypoxia (3 min, 8% O₂ 3-8 times daily for 3 days). After delivery, all animals demonstrated severe metabolic acidosis (pH 7 ± 0.12; mean ± SD) and low APGAR scores (<5 at 10 min of age). Three of 4 animals had both electrographic and clinical seizures. Serial blood samples were collected and plasma metabolites were determined by 2-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS). The 4 UCO animals and a single nonasphyxiated animal (delivered by cesarean section but without exposure to UCO or prolonged sedation) underwent brain magnetic resonance imaging (MRI) on day 8 of life. Thalamic injury was present on MRI in 3 UCO animals, but not in the control animal. Following necropsy on day 8, brain histopathology revealed neuronal injury/loss and gliosis in portions of the ventrolateral thalamus in all 4 UCO, with 2 animals also demonstrating putamen/globus pallidus involvement. In addition, all 4 UCO animals demonstrated brain stem gliosis, with neuronal loss present in the midbrain, pons, and lateral medulla in 3 of 4 animals. Transmission electron microscopy imaging of the brain tissues was performed, which demonstrated ultrastructural white matter abnormalities, characterized by perinuclear vacuolation and axonal dilation, in 3 of 4 animals. Immunolabeling of Nogo-A, a negative regulator of neuronal growth, was not increased in the injured brains compared to 2 control animals. Using GC × GC-TOFMS, we identified metabolites previously recognized as potential biomarkers of perinatal asphyxia. The basal ganglia-thalamus-brain stem injury produced by UCO is consistent with the deep nuclear/brainstem injury pattern seen in human neonates after severe, abrupt hypoxic-ischemic insults. The UCO model permits timely detection of biomarkers associated with specific patterns of neonatal brain injury, and it may ultimately be useful for validating therapeutic strategies to treat neonatal HIE.

Exploring Perinatal Asphyxia by Metabolomics

“…Juul et al extensively explored PA in a non-human primate model, namely Macaca Nemestrina, using two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) [41][42][43]. In their first work, asphyxia was induced in utero by clamping the umbilical cord blood for 15 or 18 min before delivery [41].…”

“…A set of eight metabolites were correlated to early and long-term neurodevelopmental outcomes, and four of them, namely citrate, fumarate, lactate, and propionate, were also correlated with death or cerebral palsy. In their most recent study, the authors modified their model, including some postnatal hypoxic episodes in order to mimic apneic events that occur in human newborns with HIE [43]. A protocol of intermittent hypoxia from 3 to 8 times a day for 3 days was applied.…”

“…Lactate Plasma, urine, cord blood, CSF, brain tissue, ocular tissues [25,26,29,30,32,33,35,[38][39][40][41][42][45][46][47]49,51] Succinate Plasma, urine, cord blood, brain tissue [29,30,37,38,[40][41][42][43]45,47,49,50] Alanine Plasma, urine, cord blood, CSF, brain tissue [25,26,29,30,[37][38][39][40]48,49,52] Urine [36,38,45] BCAA, Branched Chain Amino Acids.…”

Whole Blood Gene Expression Reveals Specific Transcriptome Changes in Neonatal Encephalopathy

“…One of the key reasons for this lack of neuroprotection is clinical heterogeneity [4]. For example, brain injury caused by acute or chronic hypoxia may require different therapeutic approaches [5]. Furthermore, the coexistence of perinatal infection or inflammation affects the efficacy of therapeutic interventions [4].…”

Magnetic Resonance Imaging Findings of Term and Preterm Hypoxic-Ischemic Encephalopathy: A Review of Relevant Animal Models and Correlation to Human Imaging

“…The age of the macaque used for HIE has been between 164 to 173 days of gestation with full gestation being 173 days. The brain development of the animals at this level of maturity are comparable to a late preterm infant [ 43 ]. Injury is induced through umbilical cord occlusion for 12 to 18 minutes immediately prior to delivery.…”