Abstracts

Rationale: Low intracellular chloride ([Cl-]i) is an important determinant of post-synaptic GABAA-receptor mediated inhibition. This low [Cl-]i is set by the Donnan equilibria between immobile extracellular and cytoplasmic anions, and is maintained via homeostatic [Cl-]i and volume regulation by the cation-Cl- co-transporters. However, acute brain injuries including trauma and hypoxia-ischemia alter the Donnan equilibria. This could result in cytotoxic edema, elevated [Cl-]i and proconvulsant depolarizing GABA responses. The mechanism of critical pathological changes causing post-traumatic [Cl-]i accumulation remains unknown. One possibility is that injured, gliotic areas have altered extra- and intracellular macromolecular compositions, which may change the balance of Donnan forces that set [Cl-]i and the polarity and magnitude of GABAA-receptor activity. Matrix metalloproteinases (MMPs) are involved in the degradation of the extracellular matrix. A bacterial analog, chondroitinase ABC, alters neuronal chloride, and inhibitors of MMPs 2 and 9 improve outcome after stroke. We therefore tested whether SB-3CT, a potent and selective inhibitor of MMP-2 and MMP-9, reduces neuronal [Cl-]i accumulation after acute brain injury. Methods: Acute hippocampal slices from mice expressing the Cl- sensitive fluorescent indicator Clomeleon were used as a model of severe traumatic brain injury. Two groups of slices were pre-incubated in control ACSF and in a solution containing 20 uM SB-3CT. High-resolution two-photon fluorescence imaging of neurons expressing Clomeleon was performed 0-200 um below the slice surface. Results: In both groups of slices, morphological features of acute neuronal trauma associated with the neural shear injury induced during slice preparation included swollen or shrunken cell bodies and dendritic dystrophy and varicosities, mostly in the superficial 0-50 um layer of slices. In the deep layers of the acute slices, the cell bodies of neurons were morphologically preserved and healthy. In two groups of slices, traumatized neurons were characterized by increased [Cl-]i in the superficial layers. However, statistical analysis demonstrated significantly lower [Cl-]i in the superficial layers (0-50 and 50-100 um) of slices pre-incubated in SB-SCT. At depth of 100-200 um, no significant difference was found between the two population means. Conclusions: Our results demonstrate that MMP-2 and MMP-9 inhibitor SB-3CT ameliorates trauma-induced increases in neuronal chloride that could be predicted from effects on Donnan forces. This may exert neuro-protective effects, reduce cytotoxic cerebral edema, and reduce the incidence of acute and chronic seizures after brain injury. Funding: JG (NIH/NINDS K08 1K08NS091248), KS (NIH/NINDS RO1 NS 040109)