Epileptogenic Changes in Local Network Structure Following Injury (Project 2)

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Severe traumatic brain injury (TBI) results in post-traumatic epilepsy (PTE), following a latent period of no seizures, in approximately 20% of the civilian population. The factors that influence whether a person develops epilepsy following TBI include severity of injury, patient demographic, and a wide swath of poorly characterized cellular and molecular processes that take place during the latent period. Developing effective interventional therapies to prevent PTE will depend critically on identifying the earliest molecular pathways that are specifically epileptogenic. Acquiring such data in biophysically relevant models has been technically challenging. Neuroinflammatory processes following TBI in rodents have been widely studied, but clinical translatability of findings has been poor. Studies in humans and large gyrencephalic animals have been largely confined to large- scale electrographic recordings and ex vivo physiological and histological analysis. Four findings stand out from these studies as hallmarks of an epileptic brain: 1) gliosis at the site of injury, 2) GABA-mediated synaptic activity that is excitatory rather than inhibitory, 3) a prevalence of non-ictal hypersynchronous electrical activity, and 4) disruption of functional network connectivity at the macro (whole-brain) level. Several lines of published and preliminary data suggest that gliotic tissue resulting from TBI produces an extracellular matrix that stably lowers extracellular chloride. This, in turn, depolarizes the GABA reversal potential (EGABA). We hypothesize that the resulting disinhibition will increase the synchronicity of neuronal activity, beginning at the site of injury, where the gliotic scar forms. We have recently developed a porcine model of neocortical post- traumatic epilepsy and the imaging tools (fluorophores, large-animal 2-photon microscope, and supporting technologies) to longitudinally study the epileptic focus with single-neuron precision and multimodal (chloride and calcium) fluorescence data. In this project, we will simultaneously image intra or extracellular chloride and calcium activity before injury, during the latent period, and after the emergence of spontaneous seizures. We will also chemically alter the extracellular matrix (which in turn alters extracellular chloride) to test for a causal link between extracellular chloride and functional connectivity. We will use these data to evaluate the hypothesis that depolarizing changes in EGABA produces epileptogenic changes in functional network connectivity and that these changes correlate with clinically measurable epileptic phenotypes. This rich dataset will inform development of anti-PTE treatments as follows. If a decrease in extracellular chloride is associated with increased neuronal activity and network connectivity, then treatments that alter the formation of gliotic extracellular matrix (e.g. matrix metalloproteinases) may prevent neuronal synchronization during the latent period and prevent PTE itself. If an increase in intracellular chloride is associated with increased neuronal activity and functional connectivity, then intracellular protease inhibitors or transport inhibitors may be more effective. If neuronal activity and network connectivity are uncorrelated with chloride changes, but predictive of PTE, then it may be necessary to focus on developing treatments that directly manipulate neuronal activity and functional network connectivity in injured neurons.