Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro

Abstract

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.

Inhibition of Neuronal Necroptosis Mediated by RIPK1 Provides Neuroprotective Effects on Hypoxia and Ischemia In Vitro and In Vivo

“…It was shown that at 21 DIV 59.28 ± 6.05% of cells in the hippocampal cell culture were active, the duration of calcium oscillations was 12.37 ± 1.16 s, and the frequency was 1.34 ± 0.14 osc/min ( Figure 2 A–C). Exposure to both ischemic factors leads to the inhibition of calcium activity and a significant decrease in the number of cells in which Ca 2+ events are registered (GD—41.62 ± 1.71,%; Hypoxia—35.91 ± 1.05%) and is consistent with previously published data [ 17 ]. The duration and frequency of calcium oscillations in modeling glucose deprivation and hypoxia did not change significantly.…”

“…To assess the viability of primary cultures, 7 days after modeling stress factors, cells were stained in vitro with specific fluorescent dyes: propidium iodide (Sigma Aldrich, St. Louis, MO, USA) and bisbenzimide (Sigma Aldrich, St. Louis, MO, USA), which allow visualizing the nuclei of dead cells and the total number of cells in the culture, respectively [ 17 , 28 ].…”

“…To study the features of the reorganization of the calcium dynamics of neuron-glial networks associated with the kinase blockade and the modeled ischemic factors, an original algorithm for the analysis of network characteristics of calcium activity in the AstroLab software was used [ 17 , 18 ], with a state registration certificate for computer software No. 2021612870 dated 25.02.2021.…”

“…The second protocol involved intraventricular administration at a dose of 10 μg/kg. The doses were determined based on previously published studies [ 17 ]. When intraventricular injection was performed, experimental animals were anesthetized with inhalation anesthesia (Isoflurane, Henry Schein, Great Britain).…”

Unraveling of Functional Activity of Primary Hippocampal Neuron-Glial Networks in Photodynamic Therapy Based on Tetracyanotetra(aryl)porphyrazines

“…We previously showed that primary hippocampal cultures can serve as a relevant biological model of the brain neuron-glial networks in vitro [ 24 ]. In particular, we characterized the cellular content and the features of functional activity of neuron-glial networks in the different period of cultivation in vitro [ 24 , 25 , 26 ].…”