Research Fellow Massachusetts General Hospital Charlestown, Massachusetts
Rationale: Hypoxic-ischemic encephalopathy (HIE) has remained the most common underlying pathology associated with neonatal seizures. HIE-associated seizures in neonates are often resistant to conventional anti-seizure medications such as phenobarbital and benzodiazepines (Jensen 2009, Sankar 2005). Excitatory actions of GABA in developing neurons may underlie the inefficacy of these drugs. Inhibitory or excitatory GABAergic signaling in adult or developing central nervous system is controlled by the balance between cation-chloride transporters (i.e., NKCC1 and KCC2) activity. The KCC transporters are highly expressed in mature neurons with lower [Cl-]i, while NKCC1 has its highest expression in immature neurons with higher [Cl-]i. We propose that the salt and water cotransport by these transporter families modulate neuronal response to hypoxic-ischemic injury. After injury the neuron membrane becomes more permeable to salt and water. Based on the Donnan equilibrium, KCC2-expressing neurons gain water and swell while NKCC1-expressing neurons lose water and shrink. Hence application of medications that enhance GABAergic activity in premature neurons will result in hyperexcitability rather than inhibition. Methods: We used oxygen-glucose deprivation (OGD) in organotypic hippocampal slice cultures as a model of HIE. The transgenically expressing a neuronal Cl—sensitive fluorophore (Clomeleon) slices were exposed to OGD. Slices were submerged in a chamber continuously perfused in oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF). OGD was induced, for 15 minutes, by perfusion of anoxic ACSF saturated with 95% N2 and 5% CO2, and glucose substituted with equimolar concentration of mannitol. [Cl-]i changes and neuronal volume prior, during, and after OGD were assessed in slices at days in vitro 5-13 (DIV5-13) by multi-photon imaging. Results: 1) Neurons [Cl-]i increased during OGD and remained slightly elevated two hours after OGD. 2) Assessment of neurons’ volume before and after OGD revealed a subset of neurons that shrank after OGD and a subset that swelled. 3) The neurons with lowest volume pre-OGD [Cl-]i exhibited the highest increase in [Cl-]i post-OGD. Conclusions: The hippocampal organotypic slice creates a controlled environment to study the underlying mechanisms of neuronal response to hypoxic-ischemic injury. Shrinkage or swelling of the neurons after injury indicate the role of cation-chloride transporters in neuronal response to insult. Therefore, modulation of transport activity might enhance the efficacy of antiseizure medication. Funding: Please list any funding that was received in support of this abstract.: NINDS - R35NS116852-01
