Shared inflammatory glial cell signature after stab wound injury, revealed by spatial, temporal, and cell-type-specific profiling of the murine cerebral cortex.

Traumatic brain injury leads to a highly orchestrated immune- and glial cell response partially responsible for long-lasting disability and the development of secondary neurodegenerative diseases. A holistic understanding of the mechanisms controlling the responses of specific cell types and their crosstalk is required to develop an efficient strategy for better regeneration. Here, we combine spatial and single-cell transcriptomics to chart the transcriptomic signature of the injured male murine cerebral cortex, and identify specific states of different glial cells contributing to this signature. Interestingly, distinct glial cells share a large fraction of injury-regulated genes, including inflammatory programs downstream of the innate immune-associated pathways Cxcr3 and Tlr1/2. Systemic manipulation of these pathways decreases the reactivity state of glial cells associated with poor regeneration. The functional relevance of the discovered shared signature of glial cells highlights the importance of our resource enabling comprehensive analysis of early events after brain injury.

Age-progressive interplay of HSP-proteostasis, ECM-cell junctions and biomechanics ensures C. elegans astroglial architecture.

Tissue integrity is sensitive to temperature, tension, age, and is sustained throughout life by adaptive cell-autonomous or extrinsic mechanisms. Safeguarding the remarkably-complex architectures of neurons and glia ensures age-dependent integrity of functional circuits. Here, we report mechanisms sustaining the integrity of C. elegans CEPsh astrocyte-like glia. We combine large-scale genetics with manipulation of genes, cells, and their environment, quantitative imaging of cellular/ subcellular features, tissue material properties and extracellular matrix (ECM). We identify mutants with age-progressive, environment-dependent defects in glial architecture, consequent disruption of neuronal architecture, and abnormal aging. Functional loss of epithelial Hsp70/Hsc70-cochaperone BAG2 causes ECM disruption, altered tissue biomechanics, and hypersensitivity of glia to environmental temperature and mechanics. Glial-cell junctions ensure epithelia-ECM-CEPsh glia association. Modifying glial junctions or ECM mechanics safeguards glial integrity against disrupted BAG2-proteostasis. Overall, we present a finely-regulated interplay of proteostasis-ECM and cell junctions with conserved components that ensures age-progressive robustness of glial architecture.

Dose-Related Side Effects of Intravitreal Injections of Humanized Anti-Vascular Endothelial Growth Factor in Rats: Glial Cell Reactivity and Retinal Ganglion Cell Loss.

Purpose: In a previous study, we documented that the Intravitreal injections (IVIs) of bevacizumab in rats caused a retinal inflammatory response. We now study whether the IVI of other humanized anti-VEGF: ranibizumab and aflibercept also cause an inflammatory reaction in the rat retina and if it depends on the dose administered. Finally, we study whether this reaction affects retinal ganglion cell (RGC) survival. Methods: Albino Sprague-Dawley rats received a single IVI of 5 µL of PBS or ranibizumab or aflibercept at the concentration used in clinical practice (10 µg/µL or 40 µg/µL) or at a lower concentration (0.38 µg/µL and 1.5 µg/µL) calculated to obtain within the rat eye the same concentration as in the human eye in clinical practice. Others received a single 5 µL IVI of a polyclonal goat anti-rat VEGF (0.015 µg/µL) or of vehicle (PBS). Animals were processed 7 days or 1 month later. Retinal whole mounts were immunolabeled for the detection of microglial, macroglial, RGCs, and intrinsically photosensitive RGCs (ipRGCs). Fluorescence and confocal microscopy were used to examine retinal changes, and RGCs and ipRGCs were quantified automatically or semiautomatically, respectively. Results: All the injected substances including the PBS induced detectable side effects, namely, retinal microglial cell activation and retinal astrocyte hypertrophy. However, there was a greater microglial and macroglial response when the higher concentrations of ranibizumab and aflibercept were injected than when PBS, the antibody anti-rat VEGF and the lower concentrations of ranibizumab or aflibercept were injected. The higher concentration of ranibizumab and aflibercept resulted also in significant RGC death, but did not cause appreciable ipRGC death. Conclusions: The IVI of all the substances had some retinal inflammatory effects. The IVI of humanized anti-VEGF to rats at high doses cause important side effects: severe inflammation and RGC death, but not ipRGC death.

Nigrostriatal degeneration determines dynamics of glial inflammatory and phagocytic activity.

Glial cells are key players in the initiation of innate immunity in neurodegeneration. Upon damage, they switch their basal activation state and acquire new functions in a context and time-dependent manner. Since modulation of neuroinflammation is becoming an interesting approach for the treatment of neurodegenerative diseases, it is crucial to understand the specific contribution of these cells to the inflammatory reaction and to select experimental models that recapitulate what occurs in the human disease. Previously, we have characterized a region-specific activation pattern of CD11b+ cells and astrocytes in the α-synuclein overexpression mouse model of Parkinson´s disease (PD). In this study we hypothesized that the time and the intensity of dopaminergic neuronal death would promote different glial activation states. Dopaminergic degeneration was induced with two administration regimens of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), subacute (sMPTP) and chronic (cMPTP). Our results show that in the sMPTP mouse model, the pro-inflammatory phenotype of striatal CD11b+ cells was counteracted by an anti-inflammatory astrocytic profile. In the midbrain the roles were inverted, CD11b+ cells exhibited an anti-inflammatory profile and astrocytes were pro-inflammatory. The overall response generated resulted in decreased CD4 T cell infiltration in both regions. Chronic MPTP exposure resulted in a mild and prolonged neuronal degeneration that generated a pro-inflammatory response and increased CD4 T cell infiltration in both regions. At the onset of the neurodegenerative process, microglia and astrocytes cooperated in the removal of dopaminergic terminals. With time, only microglia maintained the phagocytic activity. In the ventral midbrain, astrocytes were the main phagocytic mediators at early stages of degeneration while microglia were the major phagocytic cells in the chronic state. In this scenario, we questioned which activation pattern recapitulates better the features of glial activation in PD. Glial activation in the cMPTP mouse model reflects many pathways of their corresponding counterparts in the human brain with advanced PD. Altogether, our results point toward a context-dependent cooperativity of microglia/myeloid cells and astrocytes in response to neuronal damage and the relevance of selecting the right experimental models for the study of neuroinflammation.

Transmembrane Protein TMEM230, Regulator of Glial Cell Vascular Mimicry and Endothelial Cell Angiogenesis in High-Grade Heterogeneous Infiltrating Gliomas and Glioblastoma.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

Satellite Glial Cells in Human Disease.

Satellite glial cells (SGCs) are the main type of glial cells in sensory ganglia. Animal studies have shown that these cells play essential roles in both normal and disease states. In a large number of pain models, SGCs were activated and contributed to the pain behavior. Much less is known about SGCs in humans, but there is emerging recognition that SGCs in humans are altered in a variety of clinical states. The available data show that human SGCs share some essential features with SGCs in rodents, but many differences do exist. SGCs in DRG from patients suffering from common painful diseases, such as rheumatoid arthritis and fibromyalgia, may contribute to the pain phenotype. It was found that immunoglobulins G (IgG) from fibromyalgia patients can induce pain-like behavior in mice. Moreover, these IgGs bind preferentially to SGCs and activate them, which can sensitize the sensory neurons, causing nociception. In other human diseases, the evidence is not as direct as in fibromyalgia, but it has been found that an antibody from a patient with rheumatoid arthritis binds to mouse SGCs, which leads to the release of pronociceptive factors from them. Herpes zoster is another painful disease, and it appears that the zoster virus resides in SGCs, which acquire an abnormal morphology and may participate in the infection and pain generation. More work needs to be undertaken on SGCs in humans, and this review points to several promising avenues for better understanding disease mechanisms and developing effective pain therapies.

Human Glial Cells as Innovative Targets for the Therapy of Central Nervous System Pathologies.

In vitro and preclinical in vivo research in the last 35 years has clearly highlighted the crucial physiopathological role of glial cells, namely astrocytes/microglia/oligodendrocytes and satellite glial cells/Schwann cells in the central and peripheral nervous system, respectively. Several possible pharmacological targets to various neurodegenerative disorders and painful conditions have therefore been successfully identified, including receptors and enzymes, and mediators of neuroinflammation. However, the translation of these promising data to a clinical setting is often hampered by both technical and biological difficulties, making it necessary to perform experiments on human cells and models of the various diseases. In this review we will, therefore, summarize the most relevant data on the contribution of glial cells to human pathologies and on their possible pharmacological modulation based on data obtained in post-mortem tissues and in iPSC-derived human brain cells and organoids. The possibility of an in vivo visualization of glia reaction to neuroinflammation in patients will be also discussed.

Glia are powerhouses for sleep.

Glia take up and detoxify neurotoxic lipids on a wake-sleep cycle, in turn promoting healthy sleep.

Early Alterations in Inner-Retina Neural and Glial Saturated Responses in Lens-Induced Myopia.

Purpose: Myopic marmosets are known to exhibit significant inner retinal thinning compared to age-matched controls. The purpose of this study was to assess inner retinal activity in marmosets with lens-induced myopia compared to age-matched controls and evaluate its relationship with induced changes in refractive state and eye growth. Methods: Cycloplegic refractive error (Rx), vitreous chamber depth (VCD), and photopic full-field electroretinogram were measured in 14 marmosets treated binocularly with negative contact lenses compared to 9 untreated controls at different stages throughout the experimental period (from 74 to 369 days of age). The implicit times of the a-, b-, d-, and photopic negative response (PhNR) waves, as well as the saturated amplitude (Vmax), semi-saturation constant (K), and slope (n) estimated from intensity-response functions fitted with Naka-Rushton equations were analyzed. Results: Compared to controls, treated marmosets exhibited attenuated b-, d-, and PhNR waves Vmax amplitudes 7 to 14 days into treatment before compensatory changes in refraction and eye growth occurred. At later time points, when treated marmosets had developed axial myopia, the amplitudes and implicit times of the b-, d-, and PhNR waves were similar between groups. In controls, the PhNR wave saturated amplitude increased as the b + d-wave Vmax increased. This trend was absent in treated marmosets. Conclusions: Marmosets induced with negative defocus exhibit early alterations in inner retinal saturated amplitudes compared to controls, prior to the development of compensatory myopia. These early ERG changes are independent of refraction and eye size and may reflect early changes in bipolar, ganglion, amacrine, or glial cell physiology prior to myopia development. Translational Relevance: The early changes in retinal function identified in the negative lens-treated marmosets may serve as clinical biomarkers to help identify children at risk of developing myopia.

The Impact of Treadmill Training on Tissue Integrity, Axon Growth, and Astrocyte Modulation.

Spinal cord injury (SCI) presents a complex challenge in neurorehabilitation, demanding innovative therapeutic strategies to facilitate functional recovery. This study investigates the effects of treadmill training on SCI recovery, emphasizing motor function enhancement, neural tissue preservation, and axonal growth. Our research, conducted on a rat model, demonstrates that controlled treadmill exercises significantly improve motor functions post-SCI, as evidenced by improved scores on the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and enhanced electromyography readings. Notably, the training facilitates the preservation of spinal cord tissue, effectively reducing secondary damage and promoting the maintenance of neural fibers in the injured area. A key finding is the significant stimulation of axonal growth around the injury epicenter in trained rats, marked by increased growth-associated protein 43 (GAP43) expression. Despite these advancements, the study notes a limited impact of treadmill training on motoneuron adaptation and highlights minimal changes in the astrocyte and neuron-glial antigen 2 (NG2) response. This suggests that, while treadmill training is instrumental in functional improvements post-SCI, its influence on certain neural cell types and glial populations is constrained.

[How psychological stress aggravates intestinal inflammation]. / Hoe psychologische stress tot darmontstekingen leidt.

OBJECTIVE: To investigate the biological mechanisms underlying the associations of psychological stress and intestinal inflammation in inflammatory bowel disease (IBD). DESIGN: Experimental mouse models and large human cohorts have been used. METHOD: Consecutive mouse models with chemically induced colitis were used to investigate biological pathways though which psychological stress leads to gut inflammation. These results were validated in three human cohorts with patients with IBD. RESULTS: Stress induced elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia cells. These enteric glia cells produce the protein CSF1, that promotes monocyte accumulation in the intestinal mucosa and TNF-mediated intestinal inflammation. CONCLUSION: A pivotal role for the enteric nervous system (ENS) has been discovered in mediating the aggravating effect of psychological stress on intestinal inflammation.

Live Imaging of Migrating Neurons and Glial Progenitors Visualized by in Utero Electroporation.

During cortical development, both neurons and glial cells are generated in the germinal zone near the lateral ventricle, migrate in the correct direction, and settle in their appropriate locations. This developmental process can be clearly visualized by introducing fluorescent protein-expression vectors via in utero electroporation. In this chapter, we describe labeling methods for migrating neurons and glial progenitors, as well as methods for slice culture, and time-lapse imaging.

Reconstruction of macroglia and adult neurogenesis evolution through cross-species single-cell transcriptomic analyses.

Macroglia fulfill essential functions in the adult vertebrate brain, producing and maintaining neurons and regulating neuronal communication. However, we still know little about their emergence and diversification. We used the zebrafish D. rerio as a distant vertebrate model with moderate glial diversity as anchor to reanalyze datasets covering over 600 million years of evolution. We identify core features of adult neurogenesis and innovations in the mammalian lineage with a potential link to the rarity of radial glia-like cells in adult humans. Our results also suggest that functions associated with astrocytes originated in a multifunctional cell type fulfilling both neural stem cell and astrocytic functions before these diverged. Finally, we identify conserved elements of macroglial cell identity and function and their time of emergence during evolution.

4R-cembranoid suppresses glial cells inflammatory phenotypes and prevents hippocampal neuronal loss in LPS-treated mice.

Chronic neuroinflammation has been implicated in neurodegenerative disease pathogenesis. A key feature of neuroinflammation is neuronal loss and glial activation, including microglia and astrocytes. 4R-cembranoid (4R) is a natural compound that inhibits hippocampal pro-inflammatory cytokines and increases memory function in mice. We used the lipopolysaccharide (LPS) injection model to study the effect of 4R on neuronal density and microglia and astrocyte activation. C57BL/6J wild-type mice were injected with LPS (5 mg/kg) and 2 h later received either 4R (6 mg/kg) or vehicle. Mice were sacrificed after 72 h for analysis of brain pathology. Confocal images of brain sections immunostained for microglial, astrocyte, and neuronal markers were used to quantify cellular hippocampal phenotypes and neurons. Hippocampal lysates were used to measure the expression levels of neuronal nuclear protein (NeuN), inducible nitrous oxide synthase (iNOS), arginase-1, thrombospondin-1 (THBS1), glial cell-derived neurotrophic factor (GDNF), and orosomucoid-2 (ORM2) by western blot. iNOS and arginase-1 are widely used protein markers of pro- and anti-inflammatory microglia, respectively. GDNF promotes neuronal survival, and ORM2 and THBS1 are astrocytic proteins that regulate synaptic plasticity and inhibit microglial activation. 4R administration significantly reduced neuronal loss and the number of pro-inflammatory microglia 72 h after LPS injection. It also decreased the expression of the pro-inflammatory protein iNOS while increasing arginase-1 expression, supporting its anti-inflammatory role. The protein expression of THBS1, GDNF, and ORM2 was increased by 4R. Our data show that 4R preserves the integrity of hippocampal neurons against LPS-induced neuroinflammation in mice.

Functional synergy of a human-specific and an ape-specific metabolic regulator in human neocortex development.

Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

Experience-dependent glial pruning of synaptic glomeruli during the critical period.

Critical periods are temporally-restricted, early-life windows when sensory experience remodels synaptic connectivity to optimize environmental input. In the Drosophila juvenile brain, critical period experience drives synapse elimination, which is transiently reversible. Within olfactory sensory neuron (OSN) classes synapsing onto single projection neurons extending to brain learning/memory centers, we find glia mediate experience-dependent pruning of OSN synaptic glomeruli downstream of critical period odorant exposure. We find glial projections infiltrate brain neuropil in response to critical period experience, and use Draper (MEGF10) engulfment receptors to prune synaptic glomeruli. Downstream, we find antagonistic Basket (JNK) and Puckered (DUSP) signaling is required for the experience-dependent translocation of activated Basket into glial nuclei. Dependent on this signaling, we find critical period experience drives expression of the F-actin linking signaling scaffold Cheerio (FLNA), which is absolutely essential for the synaptic glomeruli pruning. We find Cheerio mediates experience-dependent regulation of the glial F-actin cytoskeleton for critical period remodeling. These results define a sequential pathway for experience-dependent brain synaptic glomeruli pruning in a strictly-defined critical period; input experience drives neuropil infiltration of glial projections, Draper/MEGF10 receptors activate a Basket/JNK signaling cascade for transcriptional activation, and Cheerio/FLNA induction regulates the glial actin cytoskeleton to mediate targeted synapse phagocytosis.

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

Aryl Hydrocarbon Receptor Involvement in the Sodium-Dependent Glutamate/Aspartate Transporter Regulation in Cerebellar Bergmann Glia Cells.

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, exerts its functions through the activation of specific plasma membrane receptors and transporters. Overstimulation of glutamate receptors results in neuronal cell death through a process known as excitotoxicity. A family of sodium-dependent glutamate plasma membrane transporters is responsible for the removal of glutamate from the synaptic cleft, preventing an excitotoxic insult. Glial glutamate transporters carry out more than 90% of the brain glutamate uptake activity and are responsible for glutamate recycling through the GABA/Glutamate/Glutamine shuttle. The aryl hydrocarbon receptor is a ligand-dependent transcription factor that integrates environmental clues through its ability to heterodimerize with different transcription factors. Taking into consideration the fundamental role of glial glutamate transporters in glutamatergic synapses and that these transporters are regulated at the transcriptional, translational, and localization levels in an activity-dependent fashion, in this contribution, we explored the involvement of the aryl hydrocarbon receptor, as a model of environmental integrator, in the regulation of the glial sodium-dependent glutamate/aspartate transporter. Using the model of chick cerebellar Bergmann glia cells, we report herein that the aryl hydrocarbon receptors exert a time-dependent decrease in the transporter mRNA levels and a diminution of its uptake activity. The nuclear factor kappa light chain enhancer of the activated B cell signaling pathway is involved in this regulation. Our results favor the notion of an environmentally dependent regulation of glutamate removal in glial cells and therefore strengthen the notion of the involvement of glial cells in xenobiotic neurotoxic effects.

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Hyperglycemic stress induces oxidative damage of enteric glial cells by triggering redoxosomes/p66SHC activation.

OBJECTIVES: Diabetic gastrointestinal dysfunction (DGD) is a serious complication of diabetic mellitus (DM), affecting the enteric nervous system (ENS), particular enteric glial cells (EGCs). This study aimed to elucidate the effects and underlying molecular mechanisms of hyperglycemic stress on EGCs in in vitro and in vivo models of DM. METHODS: In in vitro studies, enteric glial cell line CRL-2690 was exposed to hyperglycemia stress, and cell viability, cell apoptosis and oxidative damage were assessed. In in vivo studies, STZ-induced diabetic mice were constructed, and cell apoptosis and oxidative damage of EGCs in the duodenum of DM mice were assessed. RESULTS: The results showed that hyperglycemic stress markedly induced oxidative damage of EGCs in in vitro and in vivo models of DM. This damage was found to be dependent on the activation of redoxosomes, which involved the phosphorylation of SRC and Vav2, the up-regulation of active RAC1-GTP, and the activation of NADPH oxidase (NOX). Moreover, inhibitors of redoxosomes, such as the RAC1 inhibitor NSC23766 and the NOX inhibitor VAS2870, effectively mitigated the hyperglycemic stress-induced oxidative damage of EGCs. Additionally, inhibition of p66SHC, a downstream target of redoxosomes, attenuated oxidative damage of EGCs under hyperglycemic stress. DISCUSSION: Our findings suggest that the redoxosomes/p66SHC signaling is involved in the oxidative damage of EGCs during the pathological process of DGD. This signaling cascade may represent a potential therapeutic target for the treatment of DGD.