A plethora of neurological disorders shares a final common deadly pathway known as excitotoxicity. Among these disorders, ischemic injury is a prominent cause of death and disability worldwide. Brain ischemia stems from cardiac arrest or stroke, both responsible for insufficient blood supply to the brain parenchyma. Glucose and oxygen deficiency disrupts oxidative phosphorylation, which results in energy depletion and ionic imbalance, followed by cell membrane depolarization, calcium (Ca 2+ ) overload, and extracellular accumulation of excitatory amino acid glutamate. If tight physiological regulation fails to clear the surplus of this neurotransmitter, subsequent prolonged activation of glutamate receptors forms a vicious circle between elevated concentrations of intracellular Ca 2+ ions and aberrant glutamate release, aggravating the effect of this ischemic pathway. The activation of downstream Ca 2+ -dependent enzymes has a catastrophic impact on nervous tissue leading to cell death, accompanied by the formation of free radicals, edema, and inflammation. After decades of "neuron-centric" approaches, recent research has also finally shed some light on the role of glial cells in neurological diseases. It is becoming more and more evident that neurons and glia depend on each other. Neuronal cells, astrocytes, microglia, NG2 glia, and oligodendrocytes all have their roles in what is known as glutamate excitotoxicity. However, who is the main contributor to the ischemic pathway, and who is the unsuspecting victim? In this review article, we summarize the so-far-revealed roles of cells in the central nervous system, with particular attention to glial cells in ischemiainduced glutamate excitotoxicity, its origins, and consequences.

Highlights d RNA-seq analysis of aging, ischemic stroke, and their interaction in female mice d Response to stroke in young and aged brain is similar, but differs in magnitude d Aged ischemic brain is characterized by upregulation of type-I interferon signaling d Aged mice downregulate axonal and synaptic maintenance program after stroke

The tamoxifen-inducible Cre-loxP system is widely used to overcome gene targeting pre-adult lethality, to modify a specific cell population at desired time-points, and to visualize and trace cells in fate-mapping studies. In this study we focused on tamoxifen degradation kinetics, because for all genetic fate-mapping studies, the period during which tamoxifen or its metabolites remain active in the CNS, is essential. Additionally, we aimed to define the tamoxifen administration scheme, enabling the maximal recombination rate together with minimal animal mortality. The time window between tamoxifen injection and the beginning of experiments should be large enough to allow complete degradation of tamoxifen and its metabolites. Otherwise, these substances could promote an undesired recombination, leading to data misinterpretation. We defined the optimal time window, allowing the complete degradation of tamoxifen and its metabolites, such as 4-hydroxytamoxifen, N-desmethyltamoxifen, endoxifen and norendoxifen, in the mouse brain after intraperitoneal tamoxifen injection. We determined the biological activity of these substances in vitro, as well as a minimal effective concentration of the most potent metabolite 4-hydroxytamoxifen causing recombination in vivo. For this purpose, we analyzed the recombination rate in double transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein in NG2-expressing cells, and employed a liquid chromatography, coupled with mass spectrometry, to determine the concentration of studied substances in the brain. We determined the degradation kinetics of these substances, and revealed that this process is influenced by mouse strains, age of animals, and dosage. Our results revealed that tamoxifen and its metabolites were completely degraded within 8 days in young adult C57BL/6J mice, while the age-matched FVB/NJ male mice displayed more effective degradation. Moreover, aged C57BL/6J mice were unable to metabolize all substances within 8 days. The lowering of initial tamoxifen dose leads to a significantly faster degradation of all studied substances. A disruption of the blood-brain barrier caused no concentration changes of any tamoxifen metabolites in the ipsilateral hemisphere. Taken together, we showed that tamoxifen metabolism in mouse brains is age-, strain- and dose-dependent, and these factors should be taken into account in the experimental design.