Abstracts

Rationale: Hypoxic-ischemic (HI) injury, with an incidence of 2 to 6 of every 1,000 live births, is a type of brain injury in newborns caused by lack of oxygen and blood flow. HI injury is the leading cause of neonatal seizures. These acute seizures adversely affect the developing brain causing later handicaps including epilepsy. Current treatments with available agents such as phenobarbital, phenytoin, or benzodiazepines are only successful in controlling less than half of the neonatal seizure cases. Excitatory actions of GABA in developing neurons may underlie the inefficacy of these drugs. GABA activation causes depolarization in young neurons because they have relatively higher intracellular chloride concentration. Understanding the mechanisms of chloride regulation in immature neurons is thus crucial in development of new anti-seizure therapies.
_x000D_ Pannexin 1 (Panx1) channels have recently been reported as potential chloride channels and blocking Panx1 channels is shown to be anti-epileptic in animal models of chronic epilepsy. However, their role in acute seizures induced by acute brain injuries such as HI remains understudied. The main objective of this work is to investigate the hypothesis that blocking Panx1 channels in neonates with HI injury will limit injury induced chloride loading and GABA excitation reducing the risk of acute neonatal seizures.

Methods: We used oxygen-glucose deprivation (OGD) in organotypic hippocampal slice cultures as the in vitro model of hypoxic-ischemic brain injury. The slices are prepared from mice transgenically expressing a neuronal chloride-sensitive fluorophore (Clomeleon). Slices were submerged in a chamber continuously perfused in oxygenated (95% O₂ and 5% CO₂) media. OGD was induced, for 15 minutes, by perfusion of anoxic, glucose-free media saturated with 95% N₂ and 5% CO₂. Intracellular chloride concentration ([Cl^-]_i) and neuronal volume prior, during, and after OGD were assessed in slices at days in vitro 3-13 (DIV3-13) by multi-photon imaging. Pannexin 1 channel activity was determined using nuclear fluorescent dye. Seizure activity was determined using DC-sensitive field potential recordings via a silver/silver chloride electrode placed on top of the slice.

Results: We found that neuronal injury leads to prolonged increases in neurons intracellular chloride concentration and enhanced Panx1 channel activity. Application of Panx1 channel blockers, Mefloquine or Probenecid, limited – although did not eliminate – the injury-induced increases in intracellular chloride concentration. Panx1 blockers also reduced seizure frequency after OGD.    

Conclusions: The hippocampal organotypic slice combined with multi-photon imaging and field potential recordings create a controlled environment to study the role of Pannexin 1 channels in injury-induced changes in neuronal chloride regulation. Our findings indicate that Pannexin 1 channels play a role in generation of acute neonatal seizures. Our findings could have immediate clinical implications as the Panx1 channel blockers we used are already available and FDA-approved medication for malaria and gout.

Funding: R35 NS116852 (Kevin Staley), AES postdoctoral fellowship 2022-2023 (Fatemeh Bahari)