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—Regulation of adaptive mechanisms in the central nervous system in cerebrovascular disorders and during the development of pathological processes is an urgent problems in neurobiology and medicine. Kinases, enzymes that perform a wide range of functions in the central nervous system, are of great interest as molecular targets and possible therapeutic agents in various neurodegenerative processes and impaired energy metabolism in the nervous tissue. The vast majority of intracellular reactions that lead to activation of neuroprotective mechanisms or, conversely, cell death reactions are associated with kinase activity. Nevertheless, the functioning of kinases in pathological processes in the central nervous system is poorly understood. Here, we identified components of the neuronal kinome with previously undescribed neuroprotective properties. We evaluated cell viability and characterized the morphology of primary brain cell cultures after treatment with 34 kinase inhibitors under conditions of modeled stress (glucose deprivation). We identified several groups of neuronal kinome with different actions and characterized the most physiologically relevant kinases. It was shown that blockage of eEF2K, SRC, and IKKb (IKK2) kinases, and the structurally close associated group of kinases JAK2, CDK2/CyclinA, CDK2/CyclinE, and FLT3 maintain cell viability in primary neuronal cultures during glucose deprivation in vitro. For the several most functionally important kinases, we estimated their influence on functional calcium activity of primary neuronal cultures. The data revealed that blockage of IKKb and eEF2K kinases effectively preserves the viability of neuronal cells under glucose deprivation but does not maintain the functional calcium activity of neuron-glial networks.

The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades (SRC, Ikkb, eEF2K, and FLT4) in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.

Ischemic cerebral stroke is one of the leading causes of death and disability in humans. However, molecular processes underlying the development of this pathology remain poorly understood. There are major gaps in our understanding of metabolic changes that occur in the brain tissue during the early stages of ischemia and reperfusion. In particular, it is generally accepted that both ischemia (I) and reperfusion (R) generate reactive oxygen species (ROS) that cause oxidative stress which is one of the main drivers of the pathology, although ROS generation during I/R was never demonstrated in vivo due to the lack of suitable methods. In the present study, we record for the first time the dynamics of intracellular pH and H2O2 during I/R in cultured neurons and during experimental stroke in rats using the latest generation of genetically encoded biosensors SypHer3s and HyPer7. We detect a buildup of powerful acidosis in the brain tissue that overlaps with the ischemic core from the first seconds of pathogenesis. At the same time, no significant H2O2 generation was found in the acute phase of ischemia/reperfusion. HyPer7 oxidation in the brain was detected only 24 h later. Comparison of in vivo experiments with studies on cultured neurons under I/R demonstrates that the dynamics of metabolic processes in these models significantly differ, suggesting that a cell culture is a poor predictor of metabolic events in vivo., Highlights • The dynamics of stroke-induced changes in intracellular pH and H2O2 was recorded. • Ischemia leads to immediate acidification of the affected part of the brain. • Only slight increase in H2O2 in the brain tissue occurs during ischemia/reperfusion. • The pronounced increase in H2O2 in the brain occurs next day after I/R. • Neurons show different I/R-induced pH and H2O2 dynamics in vitro and in vivo.

We present brain imaging experiments on rat cortical areas, demonstrating that, when combined with a suitable high-brightness, cell-specific genetically encoded fluorescent marker, three-photon-excited fluorescence (3PEF), enables subcellular-resolution, cell-specific 3D brain imaging that is fully compatible and readily integrable with other nonlinear-optical imaging modalities, including two-photon-fluorescence and harmonic-generation microscopy. With laser excitation provided by sub-100-fs, 1.25-µm laser pulses, cell-specific 3PEF from astrocytes and their processes detected in parallel with a three-photon-resonance-enhanced third harmonic from blood vessels is shown to enable a high-contrast 3D imaging of gliovascular interfaces.