Abstracts

Rationale: There are currently no prophylactic treatments available to prevent post-traumatic epileptogenesis (PTE) and it is frequently medically intractable. The most common anatomical feature of PTE is astrogliosis; however, the link between gliosis and PTE remains unknown. TBI is linked to the release of matrix metalloproteinases and subsequent remodeling of the ECM. Glia that proliferate at the site of injury contribute to the formation of new ECM. Preliminary data suggest that glia form ECM that is compositionally different from mature neuronal ECM such as that found in perineuronal nets. We hypothesize that this altered ECM has more densely sulfated proteoglycans, which “crowd out” chloride in the extracellular space, decreasing inhibition and increasing excitability in the injured tissue.

Methods: Here, we directly imaged chloride in the extracellular matrix or in neuronal cytoplasm following acute TBI in 4-6 month or following chronic PTE in 16-18 month Yucatan minipigs. Pigs were chosen because their gyrencephalic brains closely approximate human anatomy as well as deformational and inertial responses to external force. We used two-photon imaging in living pigs, before and after cortical impact, to quantify the effects of acute trauma on intracellular chloride (using virally delivered chloride-sensitive fluorescent protein SuperClomeleon) and extracellular chloride (using a novel dye, ABP-dextran). We also measured chronic extracellular chloride 1 year following cortical impact or a sham procedure._x000D_ Mouse hippocampal brain slice cultures were used to test the hypothesis that sulfation of the extracellular matrix is a determinant of extracellular chloride. For this experiment, slices were incubated in ABP-dextran and imaged. They were then treated with chondroitinase-ABC, an enzyme which digests chondroitin sulfate, a key component of the ECM, and imaged again.

Results: In swine, control extracellular chloride in brain parenchyma was significantly lower than that in bulk CSF. Four hours post-injury there was a mean decrease in ABP fluorescence lifetime of 25%, corresponding to an extracellular chloride increase of approximately 30 mM. Similarly, the SClm YFP/CFP emission ratio decreased by 30%, corresponding to an increase in intracellular chloride. One year post-injury extracellular chloride was significantly lower in the injured, epileptic pig brain vs the sham, non-epileptic pig. Rodent slice cultures also exhibited parenchymal extracellular chloride that was significantly lower than that observed in bulk ACSF or culture medium. Acute treatment with chondroitinase altered extracellular chloride, supporting the hypothesis that sulfation of the ECM is correlated with extracellular chloride concentration.  

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: CURE W81XWH-15-2-0069, NIH R01NS112538, R01HD099397, R35NS116852