The type of cell death has proven to play a crucial role in cancer immunotherapy efficacy. Immunogenic cell death (ICD) enhances tumor adjuvanticity and antigenicity by releasing danger signals and altering the immune peptidome. The immunogenicity of ferroptosis, an iron-dependent form of cell death, remains uncertain. Here, we show that dendritic cell (DC) vaccines loaded with ferroptotic lysates protect mice against glioma growth, inducing IFN-γ production, and promoting robust CD8⁺ T cell infiltration, activation, and effector memory formation in the tumor microenvironment. The intrinsic immunogenicity of ferroptosis was independent of the glioma type and the ferroptosis inducer. Instead, it critically required the presence of the damage-associated molecular patterns calreticulin and ATP, rather than involving HMGB1-TLR4 signaling. However, supplementing these DAMPs into DC vaccines loaded with non-ICD lysates did not restore efficacy to the level of the ferroptosis-based DC vaccine, suggesting a more complex mechanism beyond a purely DAMP-mediated effect. These findings demonstrate that ferroptosis-loaded DC vaccines elicit a potent, tumor-specific immune response, capable of eradicating intracranial gliomas in mice, which highlights their potential in cancer immunotherapy.

Temozolomide (TMZ) resistance, driven by genetic, epigenetic, and tumor microenvironment factors, remains a significant barrier in glioblastoma (GBM) therapy. Immunogenic cell death (ICD) modalities (such as necroptosis, ferroptosis, pyroptosis) offer a promising strategy to overcome TMZ resistance by enhancing tumor immunogenicity and reducing adaptive resistance. A strategic shift from conventional TMZ-induced apoptosis to a mixed cell death approach may enhance GBM sensitivity and therapeutic outcomes. However, balancing ICD activation is critical to avoid unintended pro-tumorigenic effects. In this review, we critically evaluate TMZ’s potential to induce multiple regulated cell death modalities, including apoptosis, necroptosis, ferroptosis, pyroptosis and cuproptosis, then map the detailed crosstalk between these regulated cell death pathways and assess their ICD characteristics. From a future perspective, we discuss the challenges associated with TMZ application in GBM therapy and propose novel strategies for developing highly effective GBM therapies and improving long-term treatment outcomes.

The article is devoted to the study of the structure, physico-mechanical properties and biocompatibility of modified conduits based on bacterial cellulose (BC) to replace injured blood vessels. It has been shown that both samples have almost the same elastic recoil and are superior to synthetic vascular grafts in terms of the parameters studied. It should be noted that the first modified sample is characterized by greater elasticity and lower tensile strength compared to the second sample; however, the physico-mechanical properties of the obtained conduits are in the range corresponding to native blood vessels. Scanning electron microscopy (SEM) demonstrated that the conduits under study had a fibrillar structure with nanosized pores that enabled the adhesion of endothelial cells on the internal surface of the vascular implant, improved elasticity under transverse pressure, and raised the elasticity modulus when stretching along the fibrils. Thermogravimetry revealed that elastic recoil formation depended on the nature of polyvinyl alcohol (PVA) interaction with the nanofibrillar structure of BC rather than on the content of polyvinyl alcohol used for modification. The MTT test results confirmed no cytotoxicity and high oxygen permeability in the studied samples, opening great opportunities for their application in regenerative biomedicine to replace injured blood vessels.

Background: Modern oncology views immune system dysfunction as a key factor in carcinogenesis. The induction of immunogenic cell death (ICD), a form of regulated cell death capable of activating adaptive immunity, represents a promising therapeutic strategy. Damage-associated molecular patterns (DAMPs) play a central role in this process. This review aims to summarize current knowledge of DAMPs, their release mechanisms during ICD, their classification, and their prognostic and therapeutic significance in antitumor immunity. Methods: We systematically reviewed and synthesized literature published in Pubmed and Google Scholar on ICD and DAMPs, focusing on distinct forms of DAMPs which were categorized based on recognition mechanisms (five classes) and cellular origin (extracellular, mitochondrial, nuclear, and cytosolic). Key molecules, their receptors, downstream signaling pathways, and clinical associations were analyzed. Results: The spatiotemporally coordinated release of the pattern of DAMPs promotes dendritic cell maturation, antigen presentation, activation of cytotoxic T lymphocytes, and elimination of tumor cells. DAMPs can exhibit a dual role: they are able to induce sterile inflammation essential for antitumor immunity, but may also contribute to metastasis and chronic inflammation. Among all DAMPs, high-mobility group box 1 (HMGB1, a nuclear DAMP) and calreticulin (CRT, a cytosolic protein) demonstrate the greatest prognostic value. Other DAMPs (e.g., extracellular matrix components, uric acid) act as signal amplifiers during various forms of cell death. Conclusions: Understanding the spatiotemporal dynamics of DAMP release is critical for activating immune responses against malignant cells. Monitoring DAMPs may improve patient stratification, predict therapeutic responses, and enable personalized immunotherapeutic strategies. Further investigation of ICD mechanisms and DAMP release represents a fundamental basis for developing novel anticancer therapies.

Temozolomide (TMZ), the standard first-line chemotherapy for glioblastoma multiforme (GBM), has been a cornerstone of treatment despite its known limitations. We propose critically assessing TMZ’s potential to induce multiple regulated cell death modalities and leveraging their immunogenic properties to develop novel strategies for overcoming the resistance of GBM and enhancing its therapy.

Glioma is the most common primary brain tumor, characterized by a consistently high patient mortality rate and a dismal prognosis affecting both survival and quality of life. Substantial evidence underscores the vital role of the immune system in eradicating tumors effectively and preventing metastasis, underscoring the importance of cancer immunotherapy which could potentially address the challenges in glioma therapy. Although glioma immunotherapies have shown promise in preclinical and early-phase clinical trials, they face specific limitations and challenges that have hindered their success in further phase III trials. Resistance to therapy has been a major challenge across many experimental approaches, and as of now, no immunotherapies have been approved. In addition, there are several other limitations facing glioma immunotherapy in clinical trials, such as high intra- and inter-tumoral heterogeneity, an inherently immunosuppressive microenvironment, the unique tissue-specific interactions between the central nervous system and the peripheral immune system, the existence of the blood-brain barrier, which is a physical barrier to drug delivery, and the immunosuppressive effects of standard therapy. Therefore, in this review, we delve into several challenges that need to be addressed to achieve boosted immunotherapy against gliomas. First, we discuss the hurdles posed by the glioma microenvironment, particularly its primary cellular inhabitants, in particular tumor-associated microglia and macrophages (TAMs), and myeloid cells, which represent a significant barrier to effective immunotherapy. Here we emphasize the impact of inducing immunogenic cell death (ICD) on the migration of Th17 cells into the tumor microenvironment, converting it into an immunologically “hot” environment and enhancing the effectiveness of ongoing immunotherapy. Next, we address the challenge associated with the accurate identification and characterization of the primary immune profiles of gliomas, and their implications for patient prognosis, which can facilitate the selection of personalized treatment regimens and predict the patient’s response to immunotherapy. Finally, we explore a prospective approach to developing highly personalized vaccination strategies against gliomas, based on the search for patient-specific neoantigens. All the pertinent challenges discussed in this review will serve as a compass for future developments in immunotherapeutic strategies against gliomas, paving the way for upcoming preclinical and clinical research endeavors.

The discovery of the concept of immunogenic cell death (ICD) is a cornerstone in the development of novel anti-cancer immunotherapeutic approaches. Induction of the ICD pathway by specific anti-cancer therapeutic regimens can eliminate cancer cells by directly killing them during therapy and by activation of strong and specific anti-cancer immunity, leading to a long-lasting immunological memory that prevents cancer recurrence. ICD encompasses different forms of regulated cell death and can be triggered by many anti-cancer treatment modalities, including photodynamic therapy (PDT). PDT is a multistep procedure involving the accumulation of a light-sensitive dye known as a photosensitizer (PS) in tumor cells, followed by its activation by irradiation with a light of an appropriate wavelength. In the presence of molecular oxygen, the irradiated PS leads to the generation of cytotoxic reactive oxygen species, which can lead to ICD induction in the cancer cells. Here, we first describe in vitro methods to help optimize the PDT procedure for a specific PS. We also provide a collection of protocols and techniques for assessing ICD in vitro, including analysis of the emission of damage associated molecular patterns (DAMPs), efferocytosis, and the maturation and activation state of antigen presenting cells. Next, we describe in detail protocols for diverse tumor mouse models for assessing and characterizing ICD in vivo, such as murine tumor vaccination models. Finally, as an immunotherapeutic vaccine, we suggest using either PDT-induced dead cancer cells, preferably undergoing ICD, or dendritic cells loaded with lysates of PDT-induced cancer cells in a syngeneic orthotopic glioma model. Overall, this methodological article provides a quantitative, comprehensive set of validated tools that can be successfully used, with some adaptations, to identify, optimize and validate novel PSs in vitro and in vivo for the efficient induction of ICD during photodynamic treatment.

Targeted drug delivery for primary brain tumors, particularly gliomas, is currently a promising approach to reduce patient relapse rates. The use of substitutable scaffolds, which enable the sustained release of clinically relevant doses of anticancer medications, offers the potential to decrease the toxic burden on the patient’s organism while also enhancing their quality of life and overall survival. Upconversion nanoparticles (UCNPs) are being actively explored as promising agents for detection and monitoring of tumor growth, and as therapeutic agents that can provide isolated therapeutic effects and enhance standard chemotherapy. Our study is focused on the feasibility of constructing scaffolds using methacrylated hyaluronic acid with additional impregnation of UCNPs and the chemotherapeutic drug temozolomide (TMZ) for glioma treatment. The designed scaffolds have been demonstrated their efficacy as a drug and UCNPs delivery system for gliomas. Using the aggressive orthotopic glioma model in vivo, it was found that the scaffolds possess the capacity to ameliorate neurological disorders in mice. Moreover, upon intracranial co-implantation of the scaffolds and glioma cells, the constructs disintegrate into distinct segments, augmenting the release of UCNPs into the surrounding tissue and concurrently reducing postoperative damage to brain tissue. The use of TMZ in the scaffold composition contributed to restraining glioma development and the reduction of tumor invasiveness. Our findings unveil promising prospects for the application of photopolymerizable biocompatible scaffolds in the realm of neuro-oncology.

The formation and surface behavior of mixed monolayers and LB films of two betulin methacrylate-containing polymers with molar fraction of betulin methacrylate equal to 10

IntroductionImmunogenic cell death (ICD) has emerged as a novel option for cancer immunotherapy. The key determinants of ICD encompass antigenicity (the presence of antigens) and adjuvanticity, which involves the release of damage-associated molecular patterns (DAMPs) and various cytokines and chemokines. CX3CL1, also known as neurotactin or fractalkine, is a chemokine involved in cellular signalling and immune cell interactions. CX3CL1 has been denoted as a “find me” signal that stimulates chemotaxis of immune cells towards dying cells, facilitating efferocytosis and antigen presentation. However, in the context of ICD, it is uncertain whether CX3CL1 is an important mediator of the effects of ICD.MethodsIn this study, we investigated the intricate role of CX3CL1 in immunogenic apoptosis induced by mitoxantrone (MTX) in cancer cells. The Luminex xMAP technology was used to quantify murine cytokines, chemokines and growth factors to identify pivotal regulatory cytokines released by murine fibrosarcoma MCA205 and melanoma B16-F10 cells undergoing ICD. Moreover, a murine tumour prophylactic vaccination model was employed to analyse the effect of CX3CL1 on the activation of an adaptive immune response against MCA205 cells undergoing ICD. Furthermore, thorough analysis of the TCGA-SKCM public dataset from 98 melanoma patients revealed the role of CX3CL1 and its receptor CX3CR1 in melanoma patients.ResultsOur findings demonstrate enhanced CX3CL1 release from apoptotic MCA205 and B16-F10 cells (regardless of the cell type) but not if they are undergoing ferroptosis or accidental necrosis. Moreover, the addition of recombinant CX3CL1 to non-immunogenic doses of MTX-treated, apoptotically dying cancer cells in the murine prophylactic tumour vaccination model induced a robust immunogenic response, effectively increasing the survival of the mice. Furthermore, analysis of melanoma patient data revealed enhanced survival rates in individuals exhibiting elevated levels of CD8+ T cells expressing CX3CR1.ConclusionThese data collectively underscore the importance of the release of CX3CL1 in eliciting an immunogenic response against dying cancer cells and suggest that CX3CL1 may serve as a key switch in conferring immunogenicity to apoptosis.

Since its discovery more than 100 years ago, photodynamic therapy (PDT) has become a potent strategy for the treatment of many types of cancer [...]

Research in the past decade on immunogenic cell death (ICD) has shown that the immunogenicity of dying tumor cells is crucial for effective anticancer therapy. ICD induction leads to the emission of specific damage-associated molecular patterns (DAMPs), which act as danger signals and as adjuvants to activate specific anti-tumor immune responses, leading to the elimination of tumor cells and the formation of long-term immunological memory. ICD can be triggered by many anticancer treatment modalities, including photodynamic therapy (PDT). However, due to the variety of photosensitizers used and the lack of a universally adopted PDT protocol, there is a need to develop novel PDT with a proven ICD capability. In the present study, we characterized the abilities of two photoactive dyes to induce ICD in experimental glioma in vitro and in vivo. One dye was from the tetracyanotetra(aryl)porphyrazine group with 9-phenanthrenyl (pz I), and the other was from the 4-(4-fluorobenzyoxy)phenyl (pz III) group in the aryl frame of the macrocycle. We showed that after the photosensitizers penetrated into murine glioma GL261 cells, they localized predominantly in the Golgi apparatus and partially in the endoplasmic reticulum, providing efficient phototoxic activity against glioma GL261 cells upon light irradiation at a dose of 20 J/cm2 (λex 630 nm; 20 mW/cm2). We demonstrated that pz I-PDT and pz III-PDT can act as efficient ICD inducers when applied to glioma GL261 cells, facilitating the release of two crucial DAMPs (ATP and HMGB1). Moreover, glioma GL261 cells stimulated with pz I-PDT or pz III-PDT provided strong protection against tumor growth in a prophylactic subcutaneous glioma vaccination model. Finally, we showed that dendritic cell (DC) vaccines pulsed with the lysates of glioma GL261 cells pre-treated with pz-I-PDT or pz-III-PDT could act as effective inducers of adaptive anti-tumor immunity in an intracranial orthotopic glioma mouse model.

Sleep–wake cycle disorders most often accompany the elderly and are frequently associated with the development of neurodegenerative processes, primarily Alzheimer’s disease. Sleep disturbances can be diagnosed in patients with AD even before the onset of memory and cognitive impairment, and become more pronounced as the disease progresses. Therefore, the expansion of our knowledge of how sleep relates to AD pathogenesis needs to be addressed as soon as possible. Here, we investigated the influence of chronic sleep deprivation on the motor and orienting–exploratory activity of 5xFAD mice, as well as their spatial learning ability and long-term memory retention. The studies carried out revealed that chronic sleep deprivation negatively affects the processes of spatial memory reconsolidation in 5xFAD mice. This leads to the development of stress-related behavioral responses, including aggressive behavior. In addition, the morphological changes in the cerebral cortex, including changes in the nuclear–cytoplasmic ratio and degradation of neuronal processes are observed. Moreover, we found an increase in the level of total DNA methylation in the blood of the sleep-deprived mice, which may be one of the mechanisms of the two-way relationship between sleep and neurodegeneration.

Immunogenic cell death (ICD) arouses great interest in targeting glioma, the most common primary brain tumor, to achieve boosted immunotherapy. We discuss the unexpected findings on the induction of Th17 immunity by ICD and propose the best design for dendritic cell (DC)-based vaccines loaded with whole glioma lysates obtained after ICD inducers.

In this work we studied nanoceria (CeO2NPs) and nanoceria modified by 5-fluorouracil (5FU) as potential APIs. Nanoceria were synthesized by precipitation in a matrix of hydroxyethyl cellulose or hydroxypropylmethyl cellulose, using cerium (III) nitrate and meglumine. Nanoceria properties were estimated by UV, FTIR and X-ray photoelectron spectra; scanning electron and atomic force microscopy; powder X-ray diffraction patterns and energy dispersive X-ray microanalysis. The cytotoxicity of nanoceria and polymer-protected nanoparticles was evaluated using the established cell line NCTC clone 929 (C3H/An mouse, subcutaneous connective tissue, clone of L. line). The morphology and metabolic activity of nanoparticles at 10 μg∙mL−1 of cells was not significant. In addition, the cytotoxic effects of nanoceria were assessed on two human colorectal cancer cell lines (HT29 and HCT116), murine melanoma B16 cells and normal human skin fibroblasts. An inhibitory effect was shown for HCT116 human colorectal cancer cells. The IC50 values for pure CeO2NPs and CeO2NPs-5FU were 219.0 ± 45.6 μg∙mL−1 and 89.2 ± 14.0 μg∙mL−1, respectively. On the other hand, the IC50 of 5FU in the combination of CeO2NPs-5FU was 2-fold higher than that of pure 5FU, amounting to 5.0 nmol∙mL−1. New compositions of nanoceria modified by 5-fluorouracil in a polymer matrix were designed as a dermal polymer film and gel. The permeability of the components was studied using a Franz cell.

Immunotherapy is a proven and effective anti-tumor strategy, which can be used alongside surgery, radiation therapy, and chemotherapy [1]. Immunogenic cell death (ICD) was identified as a critical factor determining the effectiveness of cancer treatment [2]. The concept of ICD combines the capacity to destroy cancer cells effectively, with the activation of a cancer cell-specific immune response, leading to potent and long-lasting anti-cancer immunity. ICD-inducing agents activate a perilous pathway that triggers the release of ICD mediators called damage-associated molecular patterns (DAMPs). DAMPs encompass a group of naturally occurring molecules that gain immunostimulatory qualities upon exposure to the outer cell membrane or when liberated into the extracellular matrix in a specific spatiotemporal fashion. ATP, the nuclear protein HMGB1, calreticulin (CRT), and type I interferons (IFNs) are among the identified factors [8]. The concept of ICD was initially described for cancer cells undergoing apoptosis, but it was expanded to encompass additional forms of cell death, such as necroptosis, pyrroptosis, ferroptosis, nontosis, etc. [11]. Ferroptosis is a regulated iron-dependent type of cell death that is characterized by the buildup of reactive oxygen species in the cell. In this study, the immunogenicity of ferroptotic cancer cells in vitro was assessed and their potential as an alternative approach to cancer immunotherapy was tested. Glioma GL261 and fibrosarcoma MCA205 cells were induced with one of the well-known inducers of ferroptosis, RSL3 (RAS-Selective Lethal 3). After 24 hours of RSL3 stimulation, 80% of GL261 cells and 90% of MCA205 cells showed positivity to Annexin V/Sytox Blue, indicating they were in the late stage of ferroptosis. Similarly, after 3 hours of RSL3 stimulation, 50% of GL261 cells and 45% of MCA205 cells were double positive with Annexin V/Sytox Blue indicating their late-stage ferroptotic state. We evaluated the immunogenic features of early and late ferroptotic cells in vitro, specifically at 3 or 24 hours after RSL3 stimulation. To achieve this, we compared the phenotype of dendritic cells (BMDCs) exposed to late ferroptotic cells with BMDCs exposed to viable cancer cells. Furthermore, immunogenic apoptosis was induced with MTX as a positive control and LPS as a secondary positive control. Late ferroptotic MCA 205 cells surprisingly did not induce phenotypic BMDC maturation, as indicated by the lack of surface activation of costimulatory molecules CD86, CD80, and MHCII. In contrast, a less pronounced phenotypic response compared to MCA205 cells was induced by early ferroptotic glioma GL261 cells. Nonetheless, a decrease in the ability to activate dendritic cells was observed for late ferroptotic glioma cells as well. The study used the standard tumor prophylactic vaccination model on immunocompetent C57BL/6 J mice to assess the adaptive immune system activation by early ferroptotic cancer cells. Mice were immunized with early or late ferroptosis MCA205 cells. As a negative control, we used PBS or cells that underwent spontaneous necrosis. The mice that were immunized were later confronted with viable MCA205 tumor cells. Protection against tumor growth at the site of infection was deemed indicative of successful activation of the adaptive immune system. Remarkably, mice that received immunization with late ferroptotic MCA205 cells, induced with RSL3 for 24 hours, exhibited conspicuous tumor growth at the infection site, signifying that late ferroptotic cells are not immunogenic in vivo, as per our preliminary observations in vitro.

Astrocytes serve many functions in the brain related to maintaining nerve tissue homeostasis and regulating neuronal function, including synaptic transmission. It is assumed that astrocytes are crucial players in determining the physiological or pathological outcome of the brain aging process and the development of neurodegenerative diseases. Therefore, studies on the peculiarities of astrocyte physiology and interastrocytic signaling during aging are of utmost importance. Calcium waves are one of the main mechanisms of signal transmission between astrocytes, and in the present study we investigated the features of calcium dynamics in primary cultures of murine cortical astrocytes in physiological aging and hypoxia modeling in vitro. Specifically, we focused on the assessment of calcium network dynamics and the restructuring of the functional network architecture in primary astrocytic cultures. Calcium imaging was performed on days 21 ("young" astrocyte group) and 150 ("old" astrocyte group) of cultures' development in vitro. While the number of active cells and frequency of calcium events were decreased, we observed a reduced degree of correlation in calcium dynamics between neighboring cells, which was accompanied by a reduced number of functionally connected cells with fewer and slower signaling events. At the same time, an increase in the mRNA expression of anti-apoptotic factor Bcl-2 and connexin 43 was observed in "old" astrocytic cultures, which can be considered as a compensatory response of cells with a decreased level of intercellular communication. A hypoxic episode aggravates the depression of the connectivity of calcium dynamics of "young" astrocytes rather than that of "old" ones.

Glioma is the most common brain tumor, for which no significant improvement in life expectancy and quality of life is yet possible. The creation of stable fluorescent glioma cell lines is a promising tool for in-depth studies of the molecular mechanisms of glioma initialization and pathogenesis, as well as for the development of new anti-cancer strategies. Herein, a new fluorescent glioma GL261-kat cell line stably expressing a far-red fluorescent protein (TurboFP635; Katushka) was generated and characterized, and then validated in a mouse orthotopic glioma model. By using epi-fluorescence imaging, we detect the fluorescent glioma GL261-kat cells in mice starting from day 14 after the inoculation of glioma cells, and the fluorescence signal intensity increases as the glioma progresses. Tumor growth is confirmed by magnetic resonance imaging and histology. A gradual development of neurological deficit and behavioral alterations in mice is observed during glioma progression. In conclusion, our results demonstrate the significance and feasibility of using the novel glioma GL261-kat cell line as a model of glioma biology, which can be used to study the initialization of glioma and monitor its growth by lifetime non-invasive tracking of glioma cells, with the prospect of monitoring the response to anti-cancer therapy.

Brain tissue reconstruction posttraumatic injury remains a long-standing challenge in neurotransplantology, where a tissue-engineering construct (scaffold, SC) with specific biochemical properties is deemed the most essential building block. Such three-dimensional (3D) hydrogel scaffolds can be formed using brain-abundant endogenous hyaluronic acid modified with glycidyl methacrylate by employing our proprietary photopolymerisation technique. Herein, we produced 3D hyaluronic scaffolds impregnated with neurotrophic factors (BDNF, GDNF) possessing 600 kPa Young’s moduli and 336% swelling ratios. Stringent in vitro testing of fabricated scaffolds using primary hippocampal cultures revealed lack of significant cytotoxicity: the number of viable cells in the SC+BDNF (91.67 ± 1.08%) and SC+GDNF (88.69 ± 1.2%) groups was comparable to the sham values (p > 0.05). Interestingly, BDNF-loaded scaffolds promoted the stimulation of neuronal process outgrowth during the first 3 days of cultures development (day 1: 23.34 ± 1.46 µm; day 3: 37.26 ± 1.98 µm, p < 0.05, vs. sham), whereas GDNF-loaded scaffolds increased the functional activity of neuron-glial networks of cultures at later stages of cultivation (day 14) manifested in a 1.3-fold decrease in the duration coupled with a 2.4-fold increase in the frequency of Ca2+ oscillations (p < 0.05, vs. sham). In vivo studies were carried out using C57BL/6 mice with induced traumatic brain injury, followed by surgery augmented with scaffold implantation. We found positive dynamics of the morphological changes in the treated nerve tissue in the post-traumatic period, where the GDNF-loaded scaffolds indicated more favorable regenerative potential. In comparison with controls, the physiological state of the treated mice was improved manifested by the absence of severe neurological deficit, significant changes in motor and orienting-exploratory activity, and preservation of the ability to learn and retain long-term memory. Our results suggest in favor of biocompatibility of GDNF-loaded scaffolds, which provide a platform for personalized brain implants stimulating effective morphological and functional recovery of nerve tissue after traumatic brain injury.

Brain hypoxia remains an Achilles’ heel for public health that must be urgently addressed. Hypoxic damage affects both neurons and glial cells, particularly astrocytes, which are in close dynamic bi-directional communication, and are organized in plastic and tightly regulated networks. However, astroglial networks have received limited attention regarding their influence on the adaptive functional rearrangements of neural networks to oxygen deficiency. Herein, against the background of astrocytic Cx43 gap junction blockade by the selective blocker Gap19, we evaluated the features of spontaneous calcium activity and network characteristics of cells in primary cultures of the cerebral cortex, as well as the expression levels of metabotropic glutamate receptors 2 (mGluR2) and 5 (mGluR5) in the early and late periods after simulated hypoxia in vitro. We showed that, under normoxic conditions, blockade of Cx43 leads to an increase in the expression of metabotropic glutamate receptors mGluR2 and mGluR5 and long-term modulation of spontaneous calcium activity in primary cortical cultures, primarily expressed in the restructuring of the functional architectonics of neuron–glial networks through reducing the level of correlation between cells in the network and the percentage of existing correlated connections between cells. Blocking Cx43 during hypoxic injury has a pronounced neuroprotective effect. Together with the increased expression of mGluR5 receptors, a decrease in mGluR2 expression to the physiological level was found, which suggests the triggering of alternative molecular mechanisms of cell adaptation to hypoxia. Importantly, the blockade of Cx43 in hypoxic damage contributed to the maintenance of both the main parameters of the spontaneous calcium activity of primary cortical cultures and the functional architectonics of neuron–glial networks while maintaining the profile of calcium oscillations and calcium signal communications between cells at a highly correlated level. Our results demonstrate the crucial importance of astrocytic networks in functional brain adaptation to hypoxic damage and could be a promising target for the development of rational anti-hypoxic therapy.