Authors :
Presenting Author: Kieran Normoyle, MD, PhD – Massachusetts General Hospital / Harvard Medical School
Voloydymyr Dzhala, PhD – Assistant Professor, Neurology, Massachusetts General Hospital / Harvard Medical School; Kyle Lillis, PhD – Assistant Professor, Neurology, Massachusetts General Hospital / Harvard Medical School; Kiyoshi Egawa, MD, PhD – Assistant Professor, Medicine, Hokkaido University; Joseph Glykys, MD, PhD – Associate Professor, Pediatrics / Neurology, University of Iowa; Kevin Staley, MD – Division Chief, Professor, Neurology, Massachusetts General Hospital / Harvard Medical School
Rationale:
The reversal potential of GABA
A receptors (E
GABA) is dependent upon the chloride concentrations on both sides of the neuronal membrane. We recently discovered that the chloride concentration on the extracellular aspect of the membrane is non-uniform and roughly only half the chloride in bulk cerebrospinal fluid. We also observed that removal of polyanionic glycosaminoglycans from the extracellular space, mimicking injury-induced metalloprotease activation, results in a shift toward higher local extracellular chloride concentrations.
Methods:
We used 2-photon Fluorescence Lifetime Imaging (FLIM) of a custom chloride-sensitive fluorophore constrained to the extracellular space by conjugation with 10 kilodalton dextran in acute and organotypic cultures of hippocampal slices.
We used slice injury as well as 2-photon photolysis of single neurons within organotypic slices to model acute brain injuries. Intraneuronal chloride was measured with the ratiometric reporter Super Chlomeleon.
Results:
Having discovered that the extracellular chloride ([Cl
-]
o) between neurons both in vitro and in vivo is only about half that of bulk CSF chloride, and that digestion of a prominent sulfated glycosaminoglycan in the brain (chondroitin sulfate) leads to release of these sulfated moieties and a shift to higher chloride concentrations, we next asked what would happen to [Cl
-]
o when the sulfate moieties of the matrix are freed by endogenous matrix metalloproteinases (MMPs) after brain injury.
We found a strong dependence of [Cl-]o vs distance from injury, with Cl concentration increasing to the ACSF levels near the injured surface of acute slices or proximity to photolysed neurons in organotypic slices, respectively. These changes in [Cl-]o should also alter the neuronal intracellular chloride via the activity of the equilibrative high-velocity equilibrative membrane chloride transporters. We compared [Cl-]o and intracellular chloride ([Cl-]i) in each of these models, and confirmed results in vivo using cortical window implantation of adolescent mice. Finally, the release of sulfates and subsequent changes to [Cl-]o/i should be inhibited by MMP antagonists. We confirmed that broad-spectrum inhibition using the zinc chelator ZX-1 or the more specific MMP-2/9 inhibitor SB3CT also reduced the extracellular and intracellular chloride concentrations and neuronal volume after injury. These changes were evident at the cut surface of acute brain slices and in proximity to photolysed neurons.
Conclusions:
[Cl-]o is partially displaced by sulfates in the extracellular matrix. Damage to the extracellular matrix following brain injury alters the distribution of chloride in both the extra- and intracellular spaces. These findings have immediate implications for the treatment of cytotoxic edema and seizures after acute brain injury.
Funding: Neuronal Ion and Volume Shifts after Brain Injury, NINDS R35-NS116852