Search Results (401)

Adverse effects of benzodiazepines are largely mediated by GABA_A receptors containing α1-subunits. Recent efforts to replace benzodiazepines with neurosteroids are promising, but it is unclear to what extent α1-GABA_A receptors are also upregulated by neurosteroids. Here, we investigate how a prototypical benzodiazepine and neurosteroids modify a form of neuronal activity that is controlled by α1-GABA_A receptors. We compared the actions of the benzodiazepine midazolam, the naturally occurring neurosteroid allopregnanolone, and XBD173, a drug that enhances neurosteroidogenesis, on high- (HFA, >50 Hz) and low- (LFA, <50 Hz) frequency action potential activity in cultured neocortical tissue slices derived from wild-type mice via extracellular multi-unit recordings. The effects of midazolam were also quantified in slices from α2/3/5-knock-in-mice, in which the drug acts almost exclusively via α1-GABA_A receptors. In slices derived from α2/3/5-knock-in mice, low nanomolar concentrations of midazolam suppressed HFA in a selective manner. In slices derived from wild-type animals, HFA was also attenuated by midazolam. In contrast, allopregnanolone and XBD173 did not affect HFA but strongly suppressed LFA. Our findings suggest that, in contrast to benzodiazepines, neurosteroids do not dampen HFA, a type of cortical network activity tightly controlled by α1-GABA_A receptors. Full article

Full-length ACTH molecule and ACTH-related hexapeptide H-PKKRRP-OH are demonstrated by the patch-clamp method to decrease the Na_V1.8 channel activation gating system effective charge in the nociceptive neuron membrane, while ACTH-related tetrapeptide Ac-KKRR-NH₂ has no effect. ACTH(1–24), a fully functional ACTH mimetic, and H-PKKRRP-OH show analgesic effects in the formalin test in vivo. All peptides contain the cationic KKRR motif, but only H-PKKRRP-OH and ACTH(1–24) relieve acute pain, targeting the Na_V1.8 channel as a receptor. This seemingly controversial result is explained by application of conformational analysis and blind docking. Though conformational analysis indicates that both H-PKKRRP-OH and Ac-KKRR-NH₂ contain the cationic functional groups at the earlier suggested characteristic distance of 9–12 Å, Ac-KKRR-NH₂ does not interact with the S4_I voltage sensor of the Na_V1.8 channel activation gating system. The docking demonstrates that an extensive network of ligand–receptor ionic and hydrogen bonds involving D151, E157, R218, and R221 VSD_I residues, essential for the analgesic tripeptide Ac-KKK-NH₂ binding, is formed upon the H-PKKRRP-OH binding. Particularly important are the ionic bonds between the H-PKKRRP-OH C-terminal carboxylate anion and the S4_I R218 and R221 guanidinium groups. The described mechanism of Na_V1.8 channel modulation is fundamentally different from the effect of channel blockers. Full article

The development of next-generation organotypic platforms and disease models has proven crucial for the progress toward personalized therapeutic solutions in cancer. Fetal bovine serum (FBS) is a nutrient-rich cell culture supplement that contains essential factors for cell growth. However, in addition to ethical and environmental concerns, the manufacturing of tumor models requires a more standardized and controlled environment. This has led to the commercialization of several alternatives for the substitution of FBS, in the form of both animal-based and synthetic products. We here test the use of two alternatives for the culture of glioblastoma cells in the fabrication of organotypic tumor models, in combination with an insightful review of the existing literature, which allows for the elucidation of the most relevant challenges and potential solutions. We assess metabolic activity and cell proliferation in both 2D and 3D culture systems to determine the influence of serum on cell attachment and growth. The 3D culture systems are fabricated by photopolymerization of gelatin methacrylamide to achieve hydrogels that closely mimic the native tissue’s extracellular environment. We aim to advance our understanding of the role of culture media in these models and provide practical guidance to optimize experimental design and enhance reproducibility, thereby facilitating their broader adoption by the research community. These studies are key for the biofabrication of next-generation organoids and other advanced in vitro tumor models. Full article

The kynurenine pathway (KP) constitutes the primary route of tryptophan catabolism, generating a spectrum of neuroactive metabolites that profoundly influence central nervous system function. Dysregulation of the KP is increasingly recognized as a critical pathogenic mechanism underlying diverse neuropathological conditions. This review critically evaluates the most widely cited mammalian cellular models currently utilized to delineate the causal role of KP alterations in neurological disease. Specifically, this article examines primary cell cultures, immortalized and tumor-derived cell lines, stem cell-derived systems, and ex vivo organotypic brain slices and tissues, highlighting their distinct methodological advantages, translational limitations, and specific enzymatic profiles. Across the described cellular systems, a recurring mechanistic theme emerges: quinolinic acid-driven mitochondrial dysfunction, oxidative stress, and NAD+ depletion converge in neurodegenerative conditions such as Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Conversely, kynurenic acid exhibits disorder-dependent—and at times opposing—roles, attenuating dopaminergic neurotoxicity in Parkinson’s disease models while contributing to synaptic pruning deficits in schizophrenia models. Furthermore, cellular models demonstrate that IDO1/TDO induction and downstream metabolite shifts are frequently cell type- and species-dependent, complicating direct extrapolation to human pathology. Because no single experimental system achieves complete physiological fidelity, elucidating the complex dynamics of the KP and identifying novel therapeutic targets requires the integration of data across complementary platforms. Full article

Brain injury after hypoxia–ischemia (HI) is the leading cause of morbidity and mortality in term and near-term neonates worldwide. The ferret is a promising translational model to study HI due to its gyrified brain and white-to-gray matter ratio that more closely resembles humans compared to rodents. Caffeine, an adenosine A2A receptor (A2AR) antagonist, shows neuroprotective potential after HI, but its effects have not been fully characterized. We sought to evaluate caffeine’s effect on neuronal cell death, cytotoxicity, and inflammatory and oxidative stress markers in a term-equivalent ferret organotypic brain slice model of HI. Slices were cultured for 72 h, exposed to two hours of oxygen–glucose deprivation (OGD), and randomized to OGD alone, OGD with caffeine (20 or 50 mg/L), or OGD with caffeine and an A2AR agonist. Healthy slices served as controls. Outcomes included global cell death, regional cell death, microglial morphology, and expression of inflammatory and oxidative stress genes (46–48 slices/group for cell death assays and 18 slices/group for imaging, balanced by sex). Caffeine 50 mg/L significantly reduced global cell death compared to OGD (p = 0.02), and this effect persisted despite co-administration of an A2AR agonist (p = 0.01), suggesting that protection was not primarily mediated through A2AR signaling. Caffeine also did not change regional pyknotic nuclei counts (p > 0.05). Caffeine altered microglial morphology, increasing the proportion of microglia with features characteristic of control conditions. OGD significantly increased expression of inflammatory and oxidative stress-related genes (p < 0.05) compared with control slices, whereas caffeine did not significantly alter gene expression. In summary, caffeine partially reversed global cell death after OGD and altered microglial morphology. Larger, higher-powered studies are needed to further investigate caffeine’s effects on neonatal HI. Full article

Background/Objectives: Systemic inflammation and circulating proinflammatory cytokines can impair blood–brain barrier (BBB) integrity. Many in vitro BBB models lack the complexity to fully recapitulate systemic inflammation and its long-term effects on the BBB. This study aimed to develop a hollow-fiber flow-based dynamic in vitro (DIV) BBB model for investigating prolonged proinflammatory responses under physiological flow conditions. Methods: Culture conditions for hCMEC/D3 in a DIV model (Flocel) were optimized by varying serum concentrations over seven weeks. Barrier integrity (transendothelial electrical resistance (TEER), permeability studies), proliferation, metabolism (NMR spectroscopy) and molecular changes (high-throughput qPCR) were assessed. Optimized triple-cultures with hCMEC/D3, human primary astrocytes and pericytes were established. After four weeks of barrier establishment, the triple-cultures were exposed to TNF-α, IL-1β and IFN-γ (0.1 ng/mL or 10 ng/mL each) for two weeks. The inflammatory response was assessed with a multiplex cytokine array. Results: Reduced serum concentration (0.25% FBS) decreased proliferation, promoted aerobic respiration, and altered tight junction and transporter gene expression, accompanied by moderately improved barrier integrity compared with 1% or 5% FBS. In optimized triple-cultures, cytokine exposure induced concentration-dependent secretion of IL-6, IL-8, and MCP-1 and changes in mRNA levels, with minor effects on barrier integrity. Sustained cytokine release over two weeks demonstrated stable induction of inflammatory responses at the BBB. Conclusions: An organotypic DIV model of the human BBB, incorporating hCMEC/D3, human primary astrocytes and pericytes was successfully established. By enabling long-term exposure to physiologically relevant cytokine concentrations under flow conditions, this model may provide a platform to investigate functional and molecular BBB responses in inflammation-driven disease progression. Full article

Assessing autophagy may offer insights into the pathogenesis of chronic obstructive pulmonary disease (COPD). However, measuring the dynamic aspect of autophagy is challenging, and sample manipulation can cause signal fluctuations that deviate from physiological conditions. We applied an organotypic method to quantify autophagy in COPD, where it frequently demonstrates disease-related dysregulation. Blood from control and COPD participants was treated with or without chloroquine. Microtubule-associated protein 1 light chain 3B II (LC3B-II) abundance was quantified in peripheral blood mononuclear cells (PBMCs), and findings were validated by transmission electron microscopy. Our observations show that while basal LC3B-II abundance was similar between groups (p = 0.60), autophagic flux was significantly lower in the COPD cohort, suggesting disruption in the regulatory factors that direct autophagosome clearance (p = 0.004). This was supported by less frequent observations of autophagy-related vacuoles in the cytosol of COPD-derived PBMCs. Our findings indicate that the suppression of autophagy can be detected in the blood of individuals with COPD, which warrants further investigation into its contribution to extrapulmonary disease processes. Full article

Radiation-induced biological responses emerge through complex interactions across multiple biological scales, ranging from molecular damage to tissue remodeling and organism-level outcomes. Although traditional radiobiology has primarily focused on DNA damage and linear dose–response relationships, increasing evidence suggests that radiation responses are highly context-dependent and cannot be fully explained by genomic alterations alone. In particular, low-dose and chronic radiation exposures often induce biological effects that involve dynamic regulatory processes beyond direct mutational burden. The narrative review proposes a conceptual multiscale framework for predictive radiobiology that integrates genomic damage, post-transcriptional regulation, network rewiring, and tissue microenvironmental interactions. Within this framework, “predictive radiobiology” refers to the integrative prediction of radiation-induced outcomes, including radiosensitivity, tissue remodeling, fibrosis progression, therapeutic response, and long-term carcinogenic risk. We discuss how radiation-induced signaling extends beyond DNA double-strand breaks to include RNA-binding protein-mediated regulation, adaptive network responses, and extracellular matrix-dependent cellular plasticity. Recent advances in multi-omics, single-cell analysis, spatial biology, and three-dimensional organotypic models have revealed that radiation responses are governed by interconnected molecular and tissue-level processes. Furthermore, artificial intelligence and systems-level computational approaches provide new opportunities for modeling non-linear and context-dependent radiation effects across biological scales. We further discuss current limitations, including data integration challenges, reproducibility issues, and the translational gap between experimental models and clinical applications. Collectively, this conceptual framework highlights the need for integrative and multiscale approaches to improve mechanistic understanding and predictive modeling in modern radiobiology. Full article

In vitro cultured plant calli, induced through dedifferentiation, are colonized by diverse endophytes. Most of these endophytes, being substantially inherited from the mother plant and highly dependent on the host’s internal ecological niche, are termed host-dependent endophytes (HDEs). Due to their close association with their hosts, HDEs exhibit heritable characteristics. However, our current understanding of plant HDEs and their effects on the host plant is limited. In this study, we characterized the composition and potential functions of the endophytic microbiota in grapevine calli derived from different varieties and organs corresponding to Cabernet Sauvignon berry flesh (CF), Rose Honey berry flesh (RF), and Rose Honey shoot tip (RS) using high-throughput sequencing and bioinformatics. Our results showed that the genotype and organotype of the explant did not affect the alpha diversity of endophytes in callus, but were associated with differences in beta diversity and community structure of the endophytic microbiota. Different types of grapevines calli inherited distinct endophytes from their mother plants, whereas sharing a conservative core endophytic microbiota consisting of a small number of amplicon sequence variants (ASVs) with high relative abundances (bacteria: 38 ASVs ranging from 79 to 92%; fungi: 9 ASVs ranging from 32 to 58). Prediction analyses using revealed conserved functional traits of the endophytic microbiota across callus types, including a core suite of bacterial adaptive phenotypes, stable central metabolism dominated by oxidative phosphorylation, and uniformly structured fungal communities dominated by saprotrophs and pathotrophs, while consistently containing yeast-form fungi. Although minor variations such as elevated trait abundance in the CF group were noted, no statistically significant functional divergence was observed, demonstrating that the endophytic microbiota of grapevine callus maintains a conserved functional profile across different types. Collectively, this study provides a methodological framework for investigating plant HDEs and offers new insights into host-endophyte interactions at the cellular level. Full article

Palmitic acid (PA), the most abundant saturated fatty acid in the human body, is implicated in lipotoxicity under hyperlipidemic conditions, with potential consequences for bone metabolism. To investigate its impact on developing bone tissue, this study used an ex vivo organotypic embryonic chick femur model, exposing femora to control (0 µM), low (50 µM), and high (200 µM) PA concentrations. A multimodal approach, integrating microtomographic, histochemical, ultrastructural, and gene expression analyses, was used to assess tissue architecture, matrix composition, mineralization, and molecular adaptations. PA exposure significantly reduced longitudinal femoral growth, as evidenced by decreased femoral length and tissue volume. Gene expression analysis revealed reduced expression of selected osteogenic differentiation-related markers, including RUNX2, BMP2, and SPP1. However, COL1A2 expression was upregulated, correlating with increased collagenous matrix deposition and enhanced mineralization in PA-treated groups. Alcian blue staining further suggested reduced proteoglycan-rich cartilage matrix, particularly at 200 µM PA. Additionally, PA modulated the expression of both pro-inflammatory and anti-inflammatory mediators, along with increased autophagy-associated responses, as suggested by the upregulation of autophagy-related genes and the presence of autophagosomes and autolysosomes. These findings indicate that PA does not simply exert a deleterious effect on bone tissue but rather redirects the developmental trajectory of the organotypic femur by reducing longitudinal growth while promoting collagen-rich matrix maturation and mineral compaction. This response may involve altered cartilage-associated endochondral processes, fatty-acid-driven metabolic adaptation, osteoblast/osteocyte maturation, and autophagy-associated matrix processing under lipid-enriched conditions. Full article

We present a modular microfluidic platform for constructing miniaturized, compartmentalized cell culture systems that support monoculture, co-culture, and organ-on-a-chip models of human tissues. The devices provide architecturally defined three-dimensional microenvironments in which heterogeneous cell populations can be cultured in close proximity while maintaining precise spatial organization and independent access to each compartment. In vivo-like perfusion into, from, and between adjacent chambers is achieved via micro-engineered porous barriers that act as perfusion microchannels, enabling controlled convective and diffusive transport and recapitulating paracrine signaling between tissue units. As a proof of concept, we implement an adipose–immune co-culture model that reproduces key features of inflamed, insulin-resistant adipose tissue, including altered cytokine secretion and glucose uptake. Together, these features establish a versatile platform for the biofabrication of customizable single-organ and multi-organ in vitro models that more faithfully recapitulate human tissue structure and function for applications in disease modeling, immunometabolic studies, and preclinical drug testing. Full article

Microglia are brain immune cells that phagocytose cell debris and beta-amyloid plaques in patients with Alzheimer’s disease. They develop from round amoeboid cells into ramified microglia or large macrophages, which can be studied in three-dimensional organotypic mouse brain slices. In a recent publication, we showed for the first time that we can track GFAP+ astrocytes and laminin+ vessels in organotypic brain slices using live-cell imaging . The aim of the present study was to use microcontact printing on organotypic brain slices to label microglia with Iba1 and CD11b antibodies and visualise them through live-cell imaging. We show that microglia can be easily labelled with antibodies and tracked via live-cell fluorescence microscopy for up to 20 days. Incubation in lipopolysaccharide (LPS) or granulocyte–macrophage colony-stimulating factor (GM-CSF) stimulates the migration of round amoeboid microglia, whereas interleukin-10 induces their differentiation into ramified forms. Taken together, we show the first-time live cell imaging of microglia in organotypic mouse brain slices using microcontact printing. Full article

Vasculogenic mimicry (VM) is the ability of cancer stem cells (CSCs) to express an endothelial-like phenotype and participate in tumor neovascularization via the formation of a blood-conducting, matrix-rich network. We previously reported that pancreatic ductal adenocarcinoma (PDAC) CSCs develop their VM phenotype via two interacting and coordinated factors that support the formation of the VM network: (i) the overexpression of genes for endothelial factors and vascular receptors and (ii) the very high secretion of numerous pro-angiogenic/growth factors. While microenvironmental acidosis (low pHe) is an important driver of tumor metastasis, especially in PDAC, and is a component of the CSC niche, its role in VM and the ion transporters involved remains unknown. As normal stem cell differentiation is regulated by Na⁺/H⁺ exchanger 1 (NHE1)-driven pH, we investigated the role of NHE1 and the intracellular signaling involved in the acidosis-induced VM using a platform of 3D organotypic cultures composed of Matrigel with increasing concentrations of Collagen I. VM was highest on 90% Matrigel:10% Collagen I, representative of an early tumor ECM, and it decreased with increasing concentrations of Collagen I, representative of advanced tumors. In all ECM compositions, VM capacity increased stepwise with pHe acidification, and both basal and acid-stimulated VM were dependent on NHE1 activity. Acidification also decreased resting pHi and increased NHE1 proton extrusion activity, NHE1/ß1 integrin co-expression, and intracellular Ca²⁺. The stimulation of VM by extracellular acidosis depended on the transport of extracellular Ca²⁺ into the cell and the consequent increase in intracellular Ca²⁺. Altogether, these data demonstrate that extracellular acidification triggers cellular mechanisms that upregulate VM to overcome the constraints imposed by ECM composition, thereby permitting VM in ECMs where this phenotype is not expressed and extending the VM phenotype towards the tumor center to further drive metastasis. Full article

Porcine organotypic retinal explant cultures are widely used to study retinal neurodegeneration under controlled conditions, but the biological processes that occur in the retinal explant over time due to preparation-induced injury and culture are not well understood. Here, we generated a time-resolved transcriptomic reference for porcine neural retinal explants, which were maintained ex vivo for 10 days. Global expression profiles are strongly separated by culture time, with Day 0 clearly distinct from cultured samples and Day 7 and Day 10 showing the highest similarity, indicating a transition toward a later stabilized state. Across the time course, 3187 genes were differentially expressed relative to Day 0, with the largest shifts occurring at an early stage of culture (Day 1–Day 3). Pathway-level analyses revealed coordinated remodeling involving inflammatory signaling and metabolic/bioenergetic changes, including reduced mitochondrial and oxidative phosphorylation-related programs at later time points. Here, we provide a time-resolved transcriptomics reference dataset for cultured porcine retinal explants. These data can build a foundation to interpret data generated in this model, differentiate changes inherent to the explant culture from treatment-specific effects and select appropriate experimental windows for mechanistic studies of retinal degeneration. Full article

Chronic exposure to benzo[a]pyrene (BaP), a Group 1 IARC carcinogen, is a major driver of lung carcinogenesis; however, how sustained subcytotoxic exposure reprograms bronchial epithelium toward preneoplastic states remains poorly defined. Here, we subjected BEAS-2B human bronchial epithelial cells to 12 weeks of continuous BaP at environmentally relevant concentrations (0.1 and 1.0 µM) and interrogated the resulting phenotypes using an integrated multi-scale framework encompassing functional toxicology, RT-qPCR, RNA-seq, phospho-kinase/NF-κB arrays, and organotypic air–liquid interface (ALI) cultures. Cells maintained metabolic competence throughout, evidenced by sustained CYP1A1 and CYP1B1 induction at both acute (4 h) and chronic (12-week) timepoints, while accumulating genotoxic stress as demonstrated by dose-dependent nuclear γ-H2AX foci formation and ATM phosphorylation (Ser1981). RNA-seq revealed a dose-dependent transcriptional shift: 0.1 µM BaP yielded 119 differentially expressed genes (DEGs; |log2FC| ≥ 1, FDR < 0.05), whereas 1.0 µM generated 255 DEGs. Downregulated transcripts were enriched for extracellular matrix and cell-adhesion programs (COL14A1, ADAMTS2, CSMD3, CADM3), while upregulated genes encompassed inflammatory, calcium-signaling, and vesicle-trafficking modules (NFATC4, CSF2RA, SYT1, PCLO). Phospho-kinase/NF-κB arrays confirmed a p53/NF-κB signaling nexus, with concurrent activation of MAPK/ERK (Thr202/Tyr204) and PI3K/Akt (Ser473) pathways. Despite persistent genotoxic stress, cells did not acquire anchorage-independent growth and remained non-tumorigenic in vivo. Critically, ALI organotypic cultures derived from BaP-exposed cells exhibited histological dysplasia, nuclear pleomorphism, and disrupted apical-basal polarity. These findings mechanistically link chronic BaP exposure to an initiation-like preneoplastic state and establish a validated 2D/3D multi-omics platform for PAH-driven lung carcinogenesis research. Full article