Post-acute delivery of NMDA or GABAA α5 receptor antagonists promotes neurological recovery and peri-infarct brain remodeling after transient focal cerebral ischemia in mice

Ischemic stroke is the leading cause of adult disability, but drug therapies that enhance recovery remain lacking. Currently, approximately 15 million people experience a new or recurrent stroke (ischemic or hemorrhagic) worldwide yearly. Untreated ischemic stroke causes death and long-term disability in more than 20% and 70% of patients, respectively; thus, there is a compelling need for novel therapies that promote neurological recovery, brain remodeling and brain plasticity after stroke. The aim of our two studies was to investigate the effects of the delayed delivery of the N-methyl-D-aspartate (NMDA) receptor antagonist memantine and the γ-amino-butyric acid (GABAA) α5 receptor antagonist S44819 on functional neurological recovery and potential mechanisms in ischemic stroke. Therefore, we used an experimental paradigm that combines behavioral analyses in mice exposed to intraluminal transient middle cerebral artery occlusion (tMCAO) and histochemical analyses of brain remodeling and plasticity; we have previously utilized this paradigm to evaluate experimental treatments, namely, the delivery of growth factors and cell-based therapeutics. By administering two clinically applicable pharmacological compounds, i.e., memantine and S44819, we aimed to test whether our developed protocols allow for the characterization of the therapeutic effects in a more advanced translational setting. Specifically, we sought to determine whether and how the restorative effects of these two compounds differ from the effects of biological agents. The first study investigated the NMDA receptor antagonist memantine, which is widely used in human patients for the treatment of Alzheimer's disease and preferentially blocks extrasynaptic NMDA receptors, which are overactivated upon stroke and thought to disturb neuroplasticity. We hypothesized that memantine enhances post-ischemic neurological recovery and brain plasticity. C57BL6/j mice were exposed to tMCAO. Starting 72 hours post-stroke, vehicle or memantine (4 or 20 mg/kg/day) was subcutaneously delivered for 28 days. Neurological recovery, perilesional tissue remodeling and contralesional pyramidal tract plasticity were evaluated for 49 days. Memantine (20 but not 4 mg/kg/day) persistently improved motor coordination and spatial memory. Memantine reduced secondary striatal atrophy. This delayed neuroprotection was associated with reduced astrogliosis and increased capillary formation around the infarct rim. The concentrations of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) were bilaterally elevated by memantine in the striatum and cortex. The anterograde tract tracing studies revealed that memantine increased contralesional corticorubral sprouting across the midline in the direction of the ipsilesional red nucleus. In the contralesional motor cortex, the NMDA receptor subunit GluN2B, which is predominantly expressed in extrasynaptic NMDA receptors, was transiently reduced by memantine after 14 days, whereas GluN2A and post synaptic density protein 95 (PSD-95), which preferentially co-localize with synaptic NMDA receptors, were increased after 28 days. Our data support the utility of NMDA antagonists far beyond the acute stroke phase, suggesting that memantine is an attractive candidate for stroke treatment. The second study investigated whether S44819, which is a GABAA α5 receptor antagonist, promotes brain remodeling and neurological recovery during the post-acute stroke phase. GABA, which is the predominant inhibitory neurotransmitter in the adult mammalian central nervous system (CNS), plays a critical role in post-stroke neurological recovery. The tonic inhibition induced by GABA is increased in the peri-infarct cortex, resulting in cerebral hypo-excitability that compromises brain plasticity. In this study, we hypothesized that the blockade of the GABAA receptor using the selective GABAA α5 antagonist S44819 promotes brain remodeling and plasticity during the post-acute stroke phase and enhances neurological recovery. Male C57Bl6/j mice were exposed to intraluminal tMCAO. Starting at 72 hours post-stroke, vehicle or S44819 (3 or 10 mg/kg b.i.d.) was delivered p.o. for 28 days. Neurological recovery and perilesional tissue remodeling were evaluated for 42 days, i.e., up to 14 days after the completion of the S44819 delivery. S44819 (10 but not 3 mg/kg) persistently improved motor coordination and spatial memory. Secondary striatal atrophy was reduced by S44819 (10 mg/kg) 42 days post treatment (dpt), and neuronal long-term survival in the peri-infarct striatum was increased. The delayed neuroprotection was associated with reduced astrogliosis around the infarct rim 14, 28 and 42 dpt and increased brain capillary density 42 dpt. The concentrations of neurotrophic (BDNF and GDNF) and angiogenic (VEGF and fibroblast growth factors; FGF) growth factors were elevated by S44819 (10 mg/kg) in the peri-infarct, but notably not the contralesional, brain tissue 28 and 42 dpt. The GABAA α5 abundance was increased by S44819 (10 mg/kg) in the peri-infarct brain tissue 14 dpt. Contralesional pyramidal tract plasticity was not increased by S44819. Our data provide strong evidence supporting that S44819 enhances neurological recovery and perilesional brain remodeling during the post-acute stroke phase. The present study demonstrates the utility of our experimental paradigms in detecting the restorative effects of clinically applicable drugs. Notably, the GABAA α5 antagonist S44819 differed from the NMDA antagonist memantine because S44819, which reversed peri-infarct tonic inhibition, exerted its restorative actions only in the ischemic brain hemisphere, while memantine additionally elevated the growth factor levels and stimulated pyramidal tract sprouting in the contralesional hemisphere. Based on our observations, a controlled randomized phase II study, i.e., RESTORE Brain (, is currently ongoing in human stroke patients on five continents. Similar to this experimental study, the clinical study aims to evaluate the effect of S44819 delivery during the post-acute stroke phase, i.e., starting 3 days post-stoke, on functional neurological recovery as evaluated by the modified Rankin scale.



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