Blood glutamate scavengers and exercises as an effective neuroprotective treatment in mice with spinal cord injury

Abstract

OBJECTIVEExcitotoxicity due to neuronal damage and glutamate release is one of the first events that leads to the progression of neuronal degeneration and functional impairment. This study is based on a paradigm shift in the therapeutic approach for treating spinal cord injury (SCI). The authors tested a new treatment targeting removal of CNS glutamate into the blood circulation by injection of the blood glutamate scavengers (BGSs) recombinant enzyme glutamate-oxaloacetate transaminase (rGOT1) and its cosubstrate oxaloacetic acid (OxAc). Their primary objective was to investigate whether BGS treatment, followed by treadmill exercises in mice with SCI, could attenuate excitotoxicity, inflammation, scarring, and axonal degeneration and, at a later time point, improve functional recovery.METHODSA pharmacokinetic experiment was done in C57BL/6 naive mice to verify rGOT1/OxAc blood activity and to characterize the time curve of glutamate reduction in the blood up to 24 hours. The reduction of glutamate in CSF after BGS administration in mice with SCI was confirmed by high-performance liquid chromatography. Next, SCI (left hemisection) was induced in the mice, and the mice were randomly assigned to one of the following groups at 1 hour postinjury: control (underwent SCI and received PBS), treadmill exercises, rGOT1/OxAc treatment, or rGOT1/OxAc treatment followed by treadmill exercises. Treatment started 1 hour postinjury with an injection of rGOT1/OxAc and continued for 5 consecutive days. Starting 1 week after SCI, the exercises and the combined treatment groups recommenced the treadmill exercise regimen 5 days a week for 3 months. Locomotor function was assessed for 3 months using the horizontal grid walking test and CatWalk. Axonal anterograde and wallerian degenerations were evaluated using tetramethylrhodamine dextran. Tissue sections were immunofluorescently stained for Iba1, GFAP, GAP-43, synaptophysin, and NeuN.RESULTSBGS treatment decreased the CSF glutamate level up to 50%, reduced axonal wallerian degeneration, and increased axonal survival and GAP-43 expression in neuronal cells. Combined treatment reduced inflammation, scarring, and lesion size. Additionally, the combination of BGS treatment and exercises increased synapses around motor neurons and enhanced axonal regeneration through the lesion site. This resulted in motor function improvement 3 months post-SCI.CONCLUSIONSAs shown by biochemical, immunohistochemical, and functional analysis, BGSs exhibit a substantial neuroprotective effect by reducing excitotoxicity and secondary damage after SCI. Furthermore, in combination with exercises, they reduced axonal degeneration and scarring and resulted in improved functional recovery.