Ischemia–reperfusion injury (IRI) is a common phenomenon that develops both from natural causes and during major operations. Many intracellular processes mediated by calcium ions are involved in the development of IRI. Currently, chemical calcium channel blockers are used but they have a number of limitations. In this article, we study the effect of the omega-hexatoxin-Hv1a peptide toxin, an alternative to chemical calcium channel blockers, on the mechanisms of IRI development in epithelial cell culture. The toxin was produced using solid phase peptide synthesis. IRI was caused by deprivation of glucose, serum and oxygen. The data obtained demonstrate that the omega-hexatoxin-Hv1a toxin in nanomolar concentrations is able to prevent the development of apoptosis and necrosis in epithelial cells by reducing the concentration of calcium, sodium and potassium ions, as well as by delaying rapid normalization of the pH level, affecting the mitochondrial potential and oxidative stress. This toxin can be used as an alternative to chemical calcium channel blockers for preventing tissue and organ IRI due to its low-dose requirement and high bioavailability.

Background. Microglia, accounting for 5–15% of total brain cells, represent an essential population of glial cells in the cultures used for modeling neuroinflammation in vitro. However, microglia proliferation is poor in neuron–glial cultures. Here, we studied the population composition of rat hippocampal neuron–glial cell cultures prepared utilizing papain (PAP cultures) and trypsin (TRY cultures) as proteolytic enzymes for cell isolation. Methods. To evaluate the percentage and morphology of microglia in TRY and PAP cultures and cultures incubated in the presence of TGFβ+MCSF+cholesterol, which should enhance microglia proliferation, we used an immunostaining and calcium imaging approach in combination with staining using the recently developed vital microglia fluorescent probe CDr20. Results. We have shown that the microglia percentage in PAP cultures was higher than in TRY cultures. Microglia in PAP cultures are predominantly polarized, while bushy morphology was more characteristic of TRY cultures. We have also demonstrated that the TGFβ+MCSF+cholesterol combination increases the microglia number both in PAP and TRY cultures (up to 25–30%) and promotes the appearance of ameboid microglia characterized by high mobility. However, the significant appearance of ameboid microglia was observed already at the early stages of cultivation (2 DIV) in TRY cultures, while in PAP cultures, the described transformation was observed at 7 DIV. Based on the absence of the ATP-induced Ca²⁺ response, round shape, significant proliferation, and high mobility, we have suggested that ameboid microglia are reactive. Conclusions. Thus, our results demonstrate that papain is a more suitable proteolytic enzyme for preparing mixed hippocampal neuron–glial cultures with a higher percentage of heterogeneous microglia and functional neurons and astrocytes (tricultures).

The study objective. This paper reviews the express of C/D box snoRNAs (small nucleolar RNAs) and possibility of their use as biomarkers of radioresistance in chromosomal abnormalities. Material and Methods. The study compared the values of log2FC express of snoRNA C/Dbox in radiosensitive (HL-60) and radioresistant (K562) cell lines with different levels of chromosomal abnormalities. The cells were irradiated with X-ray radiation once at a dose of 4 Gy. The expression of snoRNA C/D was evaluated 1, 4 and 24 hours after irradiation, using new generation sequencing (NGS) MiSeq. Results. Different log2FC values were obtained in HL-60 and K562 cell lines 1 hour, 4 and 24 hours after irradiation. Positive expression of C/D snoRNA prevails in HL-60 throughout the experiment. In K562, the predominance of positive values of C/D snoRNA expression was observed 4 hours after irradiation, and negative values of log2FC were observed 24 hours later. the more anomalies there were in the chromosome, the greater the difference in expression we observed. at the same time, the number of C/D snoRNA changed maximally 24 hours after irradiation in the studied cell lines. We noted a greater number of C/D snoRNAs in the HL-60 cell line, and only 3 expressed C/D snoRNAs in the 15th marker chromosome in K562 out of 16 in HL-60 in the same chromosome. Conclusion. Our study showed a low informative value of using C/D snoRNAs family as markers of radiosensitivity in the presence of chromosomal abnormalities in cancer cells.

Glutamate excitotoxicity, a hallmark of acute brain injury, causes intracellular calcium overload and disrupts cation homeostasis. Although selective neuronal vulnerability is well established, the coordinated multi-ion mechanisms underlying resistance or death in distinct subpopulations, and the role of neuron–glia interactions, remain poorly understood. Using synchronous multi-channel fluorescence imaging in primary hippocampal cultures, we assessed coupled dynamics of cytosolic [Ca²⁺]i, [Na⁺]i, [K⁺]i, and pHᵢ in neurons and astrocytes exposed to toxic glutamate concentration (100 μM). Based on post-washout [Ca²⁺]ᵢ recovery, neurons were divided into fast-return, slow-return, and no-return groups. Vulnerable no-return neurons showed irreversible Ca²⁺ overload and persistent K⁺ loss, and contained the highest proportion of cells expressing calcium-permeable AMPA receptors and GluK1-containing kainate receptors. GABAergic neurons were mainly found in the slow-return and no-return groups. Fast-return neurons exhibited a two-step [Na⁺]i rise during glutamate application and washout, coinciding with a sharp [Ca²⁺]i decline, and marked cytosolic acidification; however, pHᵢ and [Na⁺]i did not recover even 40 min after washout. Their [K⁺]i recovered almost immediately and correlated inversely with [Ca²⁺]i, while [Na⁺]i correlated inversely with pHᵢ. Astrocytes rapidly cleared Ca²⁺ and stabilized pH despite sustained Na⁺ accumulation; their [K⁺]i also recovered quickly and correlated inversely with [Ca²⁺]i. GABABR agonist baclofen shifted neurons toward the slow-return group, whereas the A₁ adenosine agonist N⁶-cyclohexyladenosine abolished irreversible calcium collapse and expanded the fast-return pool. These findings clarify mechanisms of selective neuronal resilience to excitotoxicity and support Gᵢ-receptor activation as a strategy to preserve ion homeostasis.

Microglia, accounting for 5-15 % of total brain cells, is the essential population of glial cells in the cultures for modeling neuroinflammation in vitro. However, microglia proliferation is poor in the neuron-glial cultures. Here, we studied the population composition of rat hippocampal neu-ron-glial cell cultures prepared using papain (PAP-cultures) and trypsin (TRY-cultures) as prote-olytic enzymes for cell isolation. We have found that the microglia percentage in PAP-cultures was higher than in TRY-cultures. Microglia in PAP-cultures are predominantly polarized, while bushy morphology was more characterized for TRY-cultures. We have also studied the effects of the combination of TGFβ+MCSF+cholesterol, which should enhance microglia proliferation, on the population composition and microglia morphology. These factors increase the microglia number both in PAP- and TRY-cultures (to 25-30 % of the total number of cells) and promote the appearance of ameboid microglia characterized by high mobility. However, the significant appearance of ameboid microglia was observed already at the early stages of cultivation (2 DIV) in TRY-cultures while in PAP-cultures the described transformation was observed at 7DIV. Thus, our results demonstrate that papain is a more suitable proteolytic enzyme for preparing mixed hippocampal neuron-glial with a higher percentage of heterogenous microglia and functional neurons and astrocytes (tricultures).