BIOCHEMICAL AND MOLECULAR EFFECTS OF ISCHEMIA-REPERFUSION DURATION ON GLUTAMINE SYNTHETASE ACTIVITY AND NEUROPLASTICITY IN DISCRETE BRAIN REGIONS

Abstract

Despite vast and increasing promising leads from research, there are still limitations to the development of effective therapeutics for stroke. Targeting glutamate-mediated excitotoxicity through an enzyme-based approach to rescue the brain from ischemic damage may represent a propitious therapeutic strategy for stroke management. Glutamine synthetase (GS) plays an important role in the astrocytic glutamate uptake system which prevents excitotoxicity. A proper understanding of GS activity in ischemia/reperfusion (I/R) paradigms is paramount to the identification of novel treatment strategies to promote post-stroke functional recovery. Hence, this study investigated the biochemical and molecular effects of I/R duration on GS activity and neuroplasticity in discrete brain regions. Male Wistar rats were used in this study. The influence of glutamate concentration (0.1, 1.0, 10 mM), time (30, 60, 120 minutes), and brain region (cortex, striatum, cerebellum, and hippocampus) on GS activity in the brain of rats was evaluated in vitro. In the in vivo experiments, rats were subjected to bilateral common carotid artery occlusion (BCCAO) for 15 or 60 minutes to assess the effect of ischemia duration on excitatory neurochemical indices in discrete brain regions. Biochemical estimations of dopamine level, GS, glutaminase, glutamate dehydrogenase, aspartate aminotransferase, Na+ K+ ATPase, monoamine oxidase, and acetylcholinesterase activities were performed in discrete brain regions. To evaluate the effect of reperfusion duration on movement patterns and excitatory neurochemical indices in discrete brain regions, rats were subjected to BCCAO for 15 or 60 minutes followed by reperfusion for 6 or 24 hours. Animal behavior was assessed followed by biochemical evaluations in the brain. Furthermore, the effect of ischemia duration on cellular alterations in the cortex and hippocampus was assrats subjected to BCCAO for 15 or 60 minutes followed by 24 hours of reperfusion. Histological evaluation and immunohistochemical analyses for NeuN, GFAP, RIP3, and caspase 3 expressions were carried out on the cortex, dentate gyrus (DG), Cornus ammonis 1 (CA1), and Cornus ammonis 3 (CA3) of the animals. Finally, the influence of preischemia and post-ischemia GS inhibition on behavioral and cellular alterations in discrete brain regions was evaluated in rats subjected to 15 minutes of BCCAO followed by 72 hours of reperfusion. Animal behavior was assessed followed by western blot analysis, histological assessment; and immunohistochemical evaluations in the cortex and hippocampus. The results show that glutamate concentration, duration of incubation, and the brain region involved significantly influenced GS activity in vitro. Ischemia and reperfusion caused duration and brain region-dependent alterations in glutamate, glutamine, dopamine, acetylcholine, monoamine, and electrogenic homeostasis. The I/Rinduced motor deficit was occlusion and reperfusion duration dependent. Also, I/R mediated duration and brain region-dependent cellular damage in the cortex and hippocampus of rats. Moreover, pre-ischemia and post-ischemia GS inhibition by MSO ameliorated I/R-mediated behavioral and cellular changes. These results suggest that global cerebral I/R mediated loss of motor control through duration and brain region-dependent disruption of coordinated excitatory networks. In conclusion, this study shows the potential of compensatory neuroplasticity and GS inhibition as promising drug targets for stroke management and treatment.essed in