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

Ischemic brain injury is a widespread pathological condition, the main components of which are a deficiency of oxygen and energy substrates. In recent years, a number of new forms of cell death, including necroptosis, have been described. In necroptosis, a cascade of interactions between the kinases RIPK1 and RIPK3 and the MLKL protein leads to the formation of a specialized death complex called the necrosome, which triggers MLKL-mediated destruction of the cell membrane and necroptotic cell death. Necroptosis probably plays an important role in the development of ischemia/reperfusion injury and can be considered as a potential target for finding methods to correct the disruption of neural networks in ischemic damage. In the present study, we demonstrated that blockade of RIPK1 kinase by Necrostatin-1 preserved the viability of cells in primary hippocampal cultures in an in vitro model of glucose deprivation. The effect of RIPK1 blockade on the bioelectrical and metabolic calcium activity of neuron-glial networks in vitro using calcium imaging and multi-electrode arrays was assessed for the first time. RIPK1 blockade was shown to partially preserve both calcium and bioelectric activity of neuron-glial networks under ischemic factors. However, it should be noted that RIPK1 blockade does not preserve the network parameters of the collective calcium dynamics of neuron-glial networks, despite the maintenance of network bioelectrical activity (the number of bursts and the number of spikes in the bursts). To confirm the data obtained in vitro, we studied the effect of RIPK1 blockade on the resistance of small laboratory animals to in vivo modeling of hypoxia and cerebral ischemia. The use of Necrostatin-1 increases the survival rate of C57BL mice in modeling both acute hypobaric hypoxia and ischemic brain damage.

Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro.

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.

Nondestructive label-free detection of peritumoral white matter damage using cross-polarization optical coherence tomography.

Introduction: To improve the quality of brain tumor resections, it is important to differentiate zones with myelinated fibers destruction from tumor tissue and normal white matter. Optical coherence tomography (OCT) is a promising tool for brain tissue visualization and in the present study, we demonstrate the ability of cross-polarization (CP) OCT to detect damaged white matter and differentiate it from normal and tumor tissues. Materials and methods: The study was performed on 215 samples of brain tissue obtained from 57 patients with brain tumors. The analysis of the obtained OCT data included three stages: 1) visual analysis of structural OCT images; 2) quantitative assessment based on attenuation coefficients estimation in co- and cross-polarizations; 3) building of color-coded maps with subsequent visual analysis. The defining characteristics of structural CP OCT images and color-coded maps were determined for each studied tissue type, and then two classification tests were passed by 8 blinded respondents after a training. Results: Visual assessment of structural CP OCT images allows detecting white matter areas with damaged myelinated fibers and differentiate them from normal white matter and tumor tissue. Attenuation coefficients also allow distinguishing all studied brain tissue types, while it was found that damage to myelinated fibers leads to a statistically significant decrease in the values of attenuation coefficients compared to normal white matter. Nevertheless, the use of color-coded optical maps looks more promising as it combines the objectivity of optical coefficient and clarity of the visual assessment, which leads to the increase of the diagnostic accuracy of the method compared to visual analysis of structural OCT images. Conclusions: Alteration of myelinated fibers causes changes in the scattering properties of the white matter, which gets reflected in the nature of the received CP OCT signal. Visual assessment of structural CP OCT images and color-coded maps allows differentiating studied tissue types from each other, while usage of color-coded maps demonstrates higher diagnostic accuracy values in comparison with structural images (F-score = 0.85-0.86 and 0.81, respectively). Thus, the results of the study confirm the potential of using OCT as a neuronavigation tool during resections of brain tumors.

Intracellular Neuroprotective Mechanisms in Neuron-Glial Networks Mediated by Glial Cell Line-Derived Neurotrophic Factor.

Glial cell line-derived neurotrophic factor (GDNF) has a pronounced neuroprotective effect in various nervous system pathologies, including ischaemic brain damage and neurodegenerative diseases. In this work, we studied the effect of GDNF on the ultrastructure and functional activity of neuron-glial networks during acute hypoxic exposure, a key damaging factor in numerous brain pathologies. We analysed the molecular mechanisms most likely involved in the positive effects of GDNF. Hypoxia modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). GDNF (1 ng/ml) was added to the culture medium 20 min before oxygen deprivation. Acute hypoxia-induced irreversible changes in the ultrastructure of neurons and astrocytes led to the loss of functional Сa2+ activity and neural network disruption. Destructive changes in the mitochondrial apparatus and its functional activity characterized by an increase in the basal oxygen consumption rate and respiratory chain complex II activity during decreased stimulated respiration intensity were observed 24 hours after hypoxic injury. At a concentration of 1 ng/ml, GDNF maintained the functional metabolic network activity in primary hippocampal cultures and preserved the structure of the synaptic apparatus and number of mature chemical synapses, confirming its neuroprotective effect. GDNF maintained the normal structure of mitochondria in neuronal outgrowth but not in the soma. Analysis of the possible GDNF mechanism revealed that RET kinase, a component of the receptor complex, and the PI3K/Akt pathway are crucial for the neuroprotective effect of GDNF. The current study also revealed the role of GDNF in the regulation of HIF-1α transcription factor expression under hypoxic conditions.