Nearly three million people in the USA suffer traumatic brain injury (TBI) yearly; however, there are no pre- or post-TBI treatment options available. KCNQ2-5 voltage-gated K+ channels underlie the neuronal “M current”, which plays a dominant role in the regulation of neuronal excitability. Our strategy towards prevention of TBI-induced brain damage is predicated on the suggested hyper-excitability of neurons induced by TBIs, and the decrease in neuronal excitation upon pharmacological augmentation of M/KCNQ K+ currents. Seizures are very common after a TBI, making further seizures and development of epilepsy disease more likely. Our hypothesis is that TBI-induced hyperexcitability and ischemia/hypoxia lead to metabolic stress, cell death and a maladaptive inflammatory response that causes further downstream morbidity. Using the mouse controlled closed-cortical impact blunt TBI model, we found that systemic administration of the prototype M-channel “opener”, retigabine (RTG), 30 min after TBI, reduces the post-TBI cascade of events, including spontaneous seizures, enhanced susceptibility to chemo-convulsants, metabolic stress, inflammatory responses, blood–brain barrier breakdown, and cell death. This work suggests that acutely reducing neuronal excitability and energy demand via M-current enhancement may be a novel model of therapeutic intervention against post-TBI brain damage and dysfunction.

Repetitive blast traumatic brain injury (TBI) affects numerous soldiers on the battlefield. Mild TBI has been shown to have long-lasting effects with repeated injury. We have investigated effects on neuronal excitability after repetitive, mild TBI in a mouse model of blast-induced brain injury. We exposed mice to mild blast trauma of an average peak overpressure of 14.6 psi, repeated across three consecutive days. While a single exposure did not reveal trauma as indicated by the glial fibrillary acidic protein indicator, three repetitive blasts did show significant increases. As well, mice had an increased indicator of inflammation (Iba-1) and increased tau, tau phosphorylation, and altered cytokine levels in the spleen. Videoelectroencephalographic monitoring 48 h after the final blast exposure demonstrated seizures in 50% (12/24) of the mice, most of which were non-convulsive seizures. Long-term monitoring revealed that spontaneous seizures developed in at least 46% (6/13) of the mice. Patch clamp recording of dentate gyrus hippocampus neurons 48 h post-blast TBI demonstrated a shortened latency to the first spike and hyperpolarization of action potential threshold. We also found that evoked excitatory postsynaptic current amplitudes were significantly increased. These findings indicate that mild, repetitive blast exposures cause increases in neuronal excitability and seizures and eventual epilepsy development in some animals. The non-convulsive nature of the seizures suggests that subclinical seizures may occur in individuals experiencing even mild blast events, if repeated.

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Background: Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported stimulation of astrocyte mitochondrial metabolism by P2Y1 receptor agonists significantly reduced cerebral edema and reactive gliosis in a TBI model. Subsequent data on the pharmacokinetics (PK) and rapid metabolism of these compounds suggested neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. Methods: The neuroprotective efficacy of AST-004 was tested in a controlled closed cortical injury (CCCI) model of TBI in mice. Results: Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22mg/kg) exhibited significantly less secondary brain injury. These effects were quantified with less cell death (PSVue794 fluorescence) and loss of blood brain barrier breakdown (Evans Blue extravasation assay), compared to vehicle treated TBI mice. TBI treated mice also exhibited significantly reduced neuroinflammatory markers, glial-fibrillary acidic protein (GFAP, astrogliosis) and ionized Ca2+ binding adaptor molecule 1 (Iba1, microgliosis), both at the mRNA (gRT-PCR) and protein (western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, AST-004 treated TBI mice presented significantly reduced impairment of long-term memory. Spatial memory was assessed with a contextual fear conditioning behavior assay (freezing behavior after shock). Finally, AST-004 treatments were found to increase in vivo ATP production in astrocytes (gfap-targeted luciferase activity), consistent with the proposed mechanism of action. Conclusions: These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.