Abstracts

Rationale: Post-traumatic epilepsy (PTE) results from up to 20% of civilian closed head injuries. There are currently no prophylactic treatments available to prevent post-traumatic epileptogenesis and PTE is frequently medically intractable. The most common anatomical feature of brain tissue resected from patients with refractory epilepsy is gliosis. One possible connection between gliosis and PTE lies in the link between the anionic content of the extracellular matrix (ECM) and extracellular chloride concentration. Preliminary data suggest that glia proliferating at the site of injury form ECM that is compositionally different from mature neuronal ECM such as that found in perineuronal nets. This altered ECM, in turn, alters chloride in the extracellular space and in neighboring neurons. Such shifts in chloride would change the GABA reversal potential, decreasing inhibition and increasing network excitability in the injured tissue.

Methods: Here, we imaged chloride in the extracellular matrix or in neuronal cytoplasm following TBI in 4-6 month old Yucatan minipigs. Pigs have gyrencephalic brains, which closely approximate human anatomy as well as deformational and inertial responses to external force. For intraneuronal chloride imaging, animals underwent a procedure 30d prior to imaging in which bilateral burr holes were placed and corresponding durotomies performed. Each hemisphere was then injected intraparenchymally with AAV encoding the chloride sensor SuperClomeleon (SClm) under control of the synapsin promoter. The durotomy was then sealed with a transparent, flexible PDMS window. After 30d, the burr holes were exposed and SClm was imaged in both hemispheres using a custom-built large animal two-photon microscope. One hemisphere was then injured using a cortical impactor. Both hemispheres were imaged again. The animal then underwent an MRI, including diffusion weighted imaging (DWI). Finally, chloride was imaged again at a time point >4h post-injury. Animals undergoing extracellular chloride imaging followed essentially the same timeline except that burr holes and PDMS windows were placed on the day of imaging and the brain was injected bilaterally with the extracellular chloride-sensitive dye ABP-dextran. ABP was quantitatively imaged using fluorescence lifetime imaging at the same imaging time points described above.

Results: Four hours post-injury there was a mean decrease in ABP fluorescence lifetime of 25%, corresponding to an extracellular chloride increase of approximately 30mM. Similarly, the SClm YFP/CFP emission ratio decreased by 30%, corresponding to an increase in intracellular chloride. MRI DWI analysis is ongoing, but preliminary results show a mean 12% decrease in diffusivity in a subset of the lesion.

Conclusions: Together these preliminary findings are consistent with a model wherein TBI induces changes in the ECM that lead to secondary increases in both extracellular and intraneuronal chloride. If these findings are confirmed, this may point to a previously unknown mechanism of cerebral edema as well as pathological changes in GABAergic inhibition that contributes to post-traumatic epileptogenesis.

Funding: Please list any funding that was received in support of this abstract.: CURE W81XWH-15-2-0069, NIH R01NS112538, R01HD099397, R35NS116852.