Professor of Pathology, Lee J. Martin, discusses the prevalence and treatment challenges associated with hypoxic-ischemic encephalopathy (HIE), and his research that aims to identify new treatment targets such as TDP43 to improve outcomes for affected infants
Hypoxic-ischemic encephalopathy (HIE) is a brain injury caused by a reduction in oxygen and blood supply; it is the leading cause of neurodevelopmental morbidity in term infants. In babies, HIE can result from placental insufficiency, asphyxia, and cardiac arrest. In 2019, the incidence of neonatal encephalopathy was 6.5 cases/100,000 population in high-income countries, ~20 cases/100,000 population in South Asia, and ~54 cases/100,000 population in Sub-Saharan Africa.
Treatment challenges in HIE
Term HIE infants can develop bilateral (both sides) injury to brain structures, including the basal ganglia, thalamus, and cerebral cortex. HIE infants can also develop refractory seizures, and these infants have increased mortality and a greater risk of long-term severe disability and epilepsy. In Western countries, term newborns with moderate to severe HIE are treated with hypothermia (HT). With HT, babies in the neonatal intensive care units are cooled to reduce their body temperature to 4°C lower than usual for about 72 hours and then they are rewarmed to normal environment temperature. However, HT sometimes works and sometimes does not, and infants can still have variably significant impairments in cognition years later and develop cerebral palsy and persistent epilepsy.
The use of HT has been contraindicated in low to middle-income countries, and there is evidence that rewarming might trigger seizures in some settings. The effects of HT on subsequent seizure development in newborns are uncertain and merit examination. The effects of seizure on the immediate and longer-term evolution of HIE in newborns have been debated for decades and remain understudied. Better experimental insight into the mechanisms of HIE-related seizures could be leveraged to identify novel targets, including specific cells and biomolecules, and to develop new adjunctive therapeutics for HIE.
The role of interneurons
Seizures can result from too much excitation or too little inhibition, though a combination is likely. The balance of excitation and inhibition in the brain is regulated by cells called interneurons (INs). About 25 different subtypes of INs have been identified in the brains of animals and humans. Some have extravagant shapes (Fig. 1A, B) and names such as chandelier cells, bouquet cells, basket cells, Cajal-Retzius cells, and Martinotti cells. Many of these cells use the inhibitory neurotransmitter γ-aminobutyric acid (GABA), which functions to dampen excitatory activity by activating inhibitory receptors on the surface of neurons.
Some INs have robust signatures given to them by the presence of proteins called calcium-binding proteins (CBPs). Two of these CBPs are named parvalbumin (PV) and calretinin (CR) (Fig. 1A, B). Techniques using highly specific antibodies to PV and CR can be applied to thin brain sections to identify INs making PV and CR (Fig. 1A, B). They have different localization patterns in the cerebral cortex (Fig. 1A, B).
Our research on IN degeneration in HIE
We hypothesized that IN degeneration in HIE is associated with their signature CBPs, which acquire an abnormal property involving the sequestration of other key proteins that sustain DNA and RNA integrity, thereby causing single- cell death and ablation (Fig. 2). Of particular interest was TAR-DNA binding protein-43 (TDP43). TDP43 normally functions in the cell nucleus for RNA processing, cryptic exon suppression, and DNA repair. Cell inactivation of TDP43 is known to be lethal. TDP43 is already famous for its disease-causing roles in amyotrophic lateral sclerosis, Alzheimer’s disease, and frontotemporal dementia. Our lab studied seizures, INs, and TDP43 in newborn pigs because this animal better represents the human neocortex compared to rodent models and because our piglet model of HIE has been characterized extensively for its neuropathology (Martin et al., 1997; Primiani et al., 2023; Martin et al., 2025) and its associated seizure activity (Park et al., 2026).
Experimental HIE in piglets with HT treatment also translated well to human infant clinical treatment. HT is now used in many neonatal intensive care units. We discovered that IN degeneration is linked to seizure development in neonatal HIE and to TDP43 pathology. Both CR and PV INs died (Fig. 1C), as identified by the accumulation of DNA double-strand breaks using the method called TUNEL, but in different layers of the cerebral cortex. This IN death was very severe in HIE piglets without HT treatment. HT treatment rescued some of the INs. Interestingly, the CR protein became damaged at tyrosine amino acids (Fig. 2) by a toxic free radical called peroxynitrite (forming 3-nitrotyrosine). Both CR and PV formed abnormal nuclear and cytoplasmic inclusions within the INs that trapped and sequestered TDP43 (Figure 1D). This proteinopathy appeared to hinder TDP43 function because the INs accumulated DNA damage and died in a novel process that we identified as aggreosis (Fig. 2). This loss of INs strongly correlated with seizure burden.
Conclusions
IN vulnerabilities appear to be driven by their intrinsic calcium-binding protein, which abnormally traps, via protein- protein interactions, vital proteins such as TDP43. Its loss of function could cause faulty RNA processing, cryptic exon suppression, DNA repair, and DNA damage accumulation that drives IN cell death, seizures, and cerebral cortex damage in neonatal HIE (Fig. 2).
References
- Martin, L.J.; Brambrink, A.; Koehler, R.C.; Traystman, R.J. Primary sensory and forebrain motor systems in the newborn brain are preferentially damaged by hypoxia-ischemia. J. Comp. Neurol. 1997, 377(2): 262-285.
- Martin, L.J.; Lee, J.K.; Niedzwiecki, M.V.; Amrein Almira, A.; Javdan, C.; Chen, M.W.; Olberding, V.; Brown, S.M.; Park, D.; Yohannan, S.; et al. Hypothermia shifts neurodegeneration phenotype in neonatal human hypoxic-ischemic encephalopathy but not in related piglet models: possible relationship to toxic conformer and intrinsically disordered prion-like protein accumulation. Cells. 2025,14(8):586. doi:
- Primiani, C.T.; Lee, J.K.; O’Brien, C.E.; Chen, M.W.; Perin, J.; Kulikowicz, E.; Santos, P.; Adams, S.; Lester, B.; Rivera-Diaz, N.; Olberding V.; et al. Hypothermic protection in neocortex is topographic and laminar, seizure unmitigating, and partially rescues neurons depleted of RNA splicing protein Rbfox3/NeuN in neonatal hypoxic-ischemic male piglets. Cells. 2023 Oct 15;12(20):2454. doi:
- Park, D.; O’Brien, C.; Lee, J.K.; Martin, L.J. Prominent astrocytic GLAST pathology occurs in newborn human and piglet hypoxic-ischemic encephalopathy: modeling relationships among laminar neuropathology, seizures, and therapeutic hypothermia. Front. Cell. Neurosci. 2026 in press.
- Martin LJ, Ichord, RN, O’Brien CE, Yohannan S, Fernandez D, Garrido A, Amauri N, Park D, Benderoth J, Lee JK. Calretinin and parvalbumin trapping of TDP43 and XRCC1 instructs neocortical interneuron death in neonatal hypoxic-ischemic encephalopathy. Submitted for publication.
