Glial Cell Cultures Research Articles

Maternal exposure to 4-tert-octylphenol causes alterations in the morphology and function of microglia in the offspring mouse brain

4-tert-Octylphenol (OP), an endocrine disrupting chemical is widely used in the production of industrial products. Prenatal exposure to endocrine-disrupting chemicals negatively affects the brain. However, the influence of OP exposure during neurodevelopment in adult offspring remains unclear. Thus, in the present study, we investigated the effects of maternal OP exposure on brain development in adult offspring by analyzing primary glial cell cultures and mice. Our findings revealed that OP exposure led to a specific increase in the mRNA expression of the ionized calcium-binding adapter molecule 1 (Iba-1) and the proportion of amoeboid microglia in the primary glial cell culture and adult offspring mice. Exposure to OP increased the transcriptional activation of Iba-1 and estrogen response element, which were counteracted by estrogen receptor antagonists ICI 182,780. Moreover, OP exposure increased the nuclear localization of the estrogen receptor. Remarkably, OP exposure decreased the mRNA expression levels of proinflammatory cytokines and genes associated with immune response in the brains of the offspring. OP exposure upregulated actin filament-related genes and altered cytoskeletal gene expression, as demonstrated by microarray analysis. The morphological changes in microglia did not result in an inflammatory response following lipopolysaccharide treatment. Taken together, the effects of OP exposure during neurodevelopment persist into adulthood, resulting in microglial dysfunction mediated by estrogen receptor signaling pathways in the brains of adult offspring mice.

Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Ascorbic acid-containing compound efficacy in ischemic brain damage

Introduction: Ischemic brain injury remains one of the main causes of disability and mortality worldwide. Protection of cellular population, depriving from oxygen supply and nutrients, is of extreme importance for further both clinical and health outcomes of timely implemented intravascular intervention. The aim: to assess anti-ischemic activity of 3-hydroxypyridine ascorbate in the in vitro and in vivo models of brain cell and tissue response to ischemia and reoxygenation. Materials and Methods: 3-hydroxypyridine ascorbate (laboratory code 3-EA) was assessed as chemical substance (purity 99.8%) diluted in sterile phosphate-buffered saline. Intracellular Ca2+ response to glutamate excitotoxicity (GluTox), ischemia and reoxygenation as well as cellular viability was evaluated on NMRI murine fresh cortical neuro-glial cell culture incubated with 2-EA by registering intracellular Fura-2 and propidium iodide fluorescence respectively. Expression of apoptosis regulating genes BCL-2, STAT3, SOCS3, inflammation regulating genes TRAIL, MLKL, Cas-1, Cas-3, IL-1β и TNFα, and genes MAO-A and MAO-B was determined by real-time PCR. The substance neuroprotection was studied in male Sprague-Dawley rats with intraluminal middle cerebral artery (MCA) occlusion/reperfusion treated with 18 mg/kg of 2-EA along with neurological deficiency evaluation and morphological assessment of brain sections. Results: Preincubation of cortical cells with 10-100 μM of 3-EA leads to inhibition of [Ca2+]i in cytosol of neurons and astrocytes under GluTox and oxygen-glucose deprivation (OGD) conditions. Reducing [Ca2+]i inhibits necrotic cell death in an acute experiment. Incubation of cerebral cortex cells with 3-EA leads to an overexpression of anti-apoptotic genes BCL-2, STAT3, SOCS3, along with downregulation of genes TRAIL, MLKL, Cas-1, Cas-3, IL-1β and TNFα. Intraperitoneal administration of 3-EA reduces the volume of necrotic areas, perinecrotic edema, cell damage, and neurological deficits in rats with MCA occlusion. Conclusion: 3-EA dose-dependently suppresses the death of cerebral cortex cells under the excitotoxic effects of glutamate and ischemia/reoxygenation. Cell-protective effect of 3-EA involves changes in the basal and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and oxidative status proteins, which leads to inhibition of the late irreversible stages of apoptosis. A course administration of 3-hydroxypyridine ascorbate at a dose of 18 mg/kg per day reduces the severity of damage both by preserving the population of neurons in the penumbra zone and by limiting the local stress response.

Pain research in a petri dish? Advantages and limitations of neuro-glial primary cell cultures from structures of the nociceptive system

How can we learn more about pain without causing pain in humans or animals? This short review focuses on neuro-glial primary cell cultures as models to study neuro-immune interactions in the context of pain and discusses their advantages and limitations.The field of basic pain research places scientists in an ethical dilemma. We aim to understand underlying mechanisms of pain for an improved pain therapy for humans and animals. At the same time, this regularly includes the induction of pain in model animals. Within the field of psychoneuroimmunology, the examination of the complexity of neuro-immune interactions in health and disease as well as the bi-directional communication between the brain and the periphery make animal experiments an inevitable part of pain research. To address ethical and legal considerations as well as the growing societal awareness for animal welfare, scientists push for the identification and characterization of complementary methods to implement the 3R principle of Russel and Burch. As such, methods to replace animal studies, reduce the number of animals used, and refine experiments are tested. Neuro-glial primary cell cultures of structures of the nociceptive system, such as dorsal root ganglia (DRG) or the spinal dorsal horn (SDH) represent useful in vitro tools, when research comes to a cellular and molecular level. They allow for studying mechanisms of neuronal sensitization, glial cell activation, or the role of specific inflammatory mediators and intracellular signaling cascades involved in the development of inflammatory and neuropathic pain. Moreover, DRG/SDH-cultures provide the opportunity to test novel strategies for interventions, such as pharmaceuticals or cell-based therapies targeting neuroinflammatory processes. Thereby, in vitro models contribute to a better understanding of neuron-glia-immune communication in the context of pain and in the advancement of pain therapies. However, this can only be one piece in a large puzzle. Our knowledge about the complexity of pain will depend on studies in humans and animals applied in vitro and in vivo and will benefit from clear and open-minded interdisciplinary communication and transparency in public outreach.

Inflammatory Intracellular Signaling in Neurons Is Influenced by Glial Soluble Factors in iPSC-Based Cell Model of PARK2-Associated Parkinson's Disease.

Neuroinflammation is considered to be one of the driving factors in Parkinson's disease (PD). This study was conducted using neuronal and glial cell cultures differentiated from induced pluripotent stem cells (iPSC) of healthy donors (HD) and PD patients with different PARK2 mutations (PD). Based on the results of RNA sequencing, qPCR and ELISA, we revealed transcriptional and post-transcriptional changes in HD and PD neurons cultivated in HD and PD glial-conditioned medium. We demonstrated that if one or both of the components of the system, neurons or glia, is Parkin-deficient, the interaction resulted in the down-regulation of a number of key genes related to inflammatory intracellular pathways and negative regulation of apoptosis in neurons, which might be neuroprotective. In PD neurons, the stress-induced up-regulation of APLNR was significantly stronger compared to HD neurons and was diminished by glial soluble factors, both HD and PD. PD neurons in PD glial conditioned medium increased APLN expression and also up-regulated apelin synthesis and release into intracellular fluid, which represented another compensatory action. Overall, the reported results indicate that neuronal self-defense mechanisms contribute to cell survival, which might be characteristic of PD patients with Parkin-deficiency.

Multifaceted interactions between cancer cells and glial cells in brain metastasis.

Cancer brain metastasis has a poor prognosis, is commonly observed in clinical practice, and the number of cases is increasing as overall cancer survival improves. However, experiments in mouse models have shown that brain metastasis itself is an inefficient process. One reason for this inefficiency is the brain microenvironment, which differs significantly from that of other organs, making it difficult for cancer cells to adapt. The brain microenvironment consists of unique resident cell types such as neurons, oligodendrocytes, astrocytes, and microglia. Accumulating evidence over the past decades suggests that the interactions between cancer cells and glial cells can positively or negatively influence the development of brain metastasis. Nevertheless, elucidating the complex interactions between cancer cells and glial cells remains challenging, in part due to the limitations of existing experimental models for glial cell culture. In this review, we first provide an overview of glial cell culture methods and then examine recent discoveries regarding the interactions between brain metastatic cancer cells and the surrounding glial cells, with a special focus on astrocytes and microglia. Finally, we discuss future perspectives for understanding the multifaceted interactions between cancer cells and glial cells for the treatment of metastatic brain tumors.

Modification of Gas6 Protein in the Brain by a Functional Endogenous Tissue Vitamin K Cycle.

The TAM receptor ligand Gas6 is known for regulating inflammatory and immune pathways in various organs including the brain. Gas6 becomes fully functional through the post-translational modification of multiple glutamic acid residues into γ-carboxyglutamic in a vitamin K-dependent manner. However, the significance of this mechanism in the brain is not known. We report here the endogenous expression of multiple components of the vitamin K cycle within the mouse brain at various ages as well as in distinct brain glial cells. The brain expression of all genes was increased in the postnatal ages, mirroring their profiles in the liver. In microglia, the proinflammatory agent lipopolysaccharide caused the downregulation of all key vitamin K cycle genes. A secreted Gas6 protein was detected in the medium of both mouse cerebellar slices and brain glial cell cultures. Furthermore, the endogenous Gas6 γ-carboxylation level was abolished through incubation with the vitamin K antagonist warfarin and could be restored through co-incubation with vitamin K1. Finally, the γ-carboxylation level of the Gas6 protein within the brains of warfarin-treated rats was found to be significantly reduced ex vivo compared to the control brains. In conclusion, we demonstrated for the first time the existence of a functional vitamin K cycle within rodent brains, which regulates the functional modification of endogenous brain Gas6. These results indicate that vitamin K is an important nutrient for the brain. Furthermore, the measurement of vitamin K-dependent Gas6 functionality could be an indicator of homeostatic or disease mechanisms in the brain, such as in neurological disorders where Gas6/TAM signalling is impaired.

Isolation of Myenteric and Submucosal Plexus from Mouse Gastrointestinal Tract and Subsequent Co-Culture with Small Intestinal Organoids.

Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle cells, the immune system, and the microbiota. The disruption of this balance underpins the onset of gastrointestinal-related diseases. The scarcity of models replicating the intricate interplay between the ENS and the intestinal epithelium highlights the imperative for developing novel methods. We have pioneered a sophisticated tridimensional in vitro technique, coculturing small intestinal organoids with myenteric and submucosal neurons. Notably, we have made significant advances in (1) refining the isolation technique for culturing the myenteric plexus, (2) enhancing the isolation of the submucosal plexus-both yielding mixed cultures of enteric neurons and glial cells from both plexuses, and (3) subsequently co-culturing myenteric and submucosal neurons with small intestinal organoids. This co-culture system establishes neural innervations with intestinal organoids, allowing for the investigation of regulatory interactions in the context of gastrointestinal diseases. Furthermore, we have developed a method for microinjecting the luminal space of small intestinal organoids with fluorescently labeled compounds. This technique possesses broad applicability such as the assessment of intestinal permeability, transcytosis, and immunocytochemical and immunofluorescence applications. This microinjection method could be extended to alternative experimental setups, incorporating bacterial species, or applying treatments to study ENS-small intestinal epithelium interactions. Therefore, this technique serves as a valuable tool for evaluating the intricate interplay between neuronal and intestinal epithelial cells (IECs) and shows great potential for drug screening, gene editing, the development of novel therapies, the modeling of infectious diseases, and significant advances in regenerative medicine. The co-culture establishment process spans twelve days, making it a powerful asset for comprehensive research in this critical field.

INFLUENCE OF FIBROBLAST GROWTH FACTOR-2 ON THE ACTIVATION OF MOUSE HIPPOCAMPAL MICROGLIA IN A LIPOPOLYSACCHARIDE-INDUCED NEUROINFLAMMATION MODEL IN VITRO

Introduction. Activation of the pro-inflammatory phenotype of microglia may be one of the causes of chronic neuroinflammation and, as a result, can lead to pathological conditions of the brain. The use of neurotrophic factors, such as FGF2, may become one of the promising methods for correcting neurodegenerative diseases, but the question of the influence of this factor on microglia activation is still open. The purpose of this work was to study the effect of FGF2 on the activation of mouse hippocampal microglia in an in vitro model of neuroinflammation induced by lipopolysaccharide. Material and methods. The study was carried out on a culture of primary mixed glial cells of the mouse hippocampus. To quantify, area, and morphological changes in microglial cells in response to the effects of LPS and FGF2, immunocytochemical analysis was performed for markers of astrocytes (GFAP) and microglia (Iba-1). Analysis of the expression level of IL-1β, IL-6, IL-10, TNF-α was carried out by quantitative PCR. Results. In response to LPS exposure, there was an increase in the percentage of Iba-1+ cells, their surface area, changes in morphological characteristics, together with an increase in the level of expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and anti-inflammatory cytokine IL-10 both within 6, so 12 hours of exposition. The addition of FGF2 led to a decrease in the percentage of Iba-1+ cells in culture and a decrease in the surface area of microglia. A decrease in the relative level of expression of IL-1β and IL-6 was noted in the groups where LPS and FGF2 were added, while the expression of TNF-α did not change. With an increase in cultivation time up to 12 hours, an increased expression of IL-10 was detected in this group. Conclusion. Stimulation with LPS promotes the transition of microglia from a resting to an activated pro-inflammatory phenotype, as evidenced by increased proliferative activity of Iba-1+ cells in combination with an increase in the level of expression of pro-inflammatory cytokines. Inhibition of two of the three pro-inflammatory cytokines (IL-1β and IL-6) and a significant increase in the level of IL-10 in cell culture suggests an anti-inflammatory effect of FGF2.

Impact of calcitriol and PGD2-G-loaded lipid nanocapsules on oligodendrocyte progenitor cell differentiation and remyelination.

Multiple sclerosis (MS) is a demyelinating and inflammatory disease of the central nervous system (CNS) in need of a curative treatment. MS research has recently focused on the development of pro-remyelinating treatments and neuroprotective therapies. Here, we aimed at favoring remyelination and reducing neuro-inflammation in a cuprizone mouse model of brain demyelination using nanomedicines. We have selected lipid nanocapsules (LNC) coated with the cell-penetrating peptide transactivator of translation (TAT), loaded with either a pro-remyelinating compound, calcitriol (Cal-LNC TAT), or an anti-inflammatory bioactive lipid, prostaglandin D2-glycerol ester (PGD2-G) (PGD2-G-LNC TAT). Following the characterization of these formulations, we showed that Cal-LNC TAT in combination with PGD2-G-LNC TAT increased the mRNA expression of oligodendrocyte differentiation markers both in the CG-4 cell line and in primary mixed glial cell (MGC) cultures. However, while the combination of Cal-LNC TAT and PGD2-G-LNC TAT showed promising results in vitro, no significant impact, in terms of remyelination, astrogliosis, and microgliosis, was observed in vivo in the corpus callosum of cuprizone-treated mice following intranasal administration. Thus, although calcitriol's beneficial effects have been abundantly described in the literature in the context of MS, here, we show that the different doses of calcitriol tested had a negative impact on the mice well-being and showed no beneficial effect in the cuprizone model in terms of remyelination and neuro-inflammation, alone and when combined with PGD2-G-LNC TAT.

In vitro biocompatibility evaluation of functional electrically stimulating microelectrodes on primary glia.

Neural interfacing devices interact with the central nervous system to alleviate functional deficits arising from disease or injury. This often entails the use of invasive microelectrode implants that elicit inflammatory responses from glial cells and leads to loss of device function. Previous work focused on improving implant biocompatibility by modifying electrode composition; here, we investigated the direct effects of electrical stimulation on glial cells at the electrode interface. A high-throughput in vitro system that assesses primary glial cell response to biphasic stimulation waveforms at 0mA, 0.15mA, and 1.5mA was developed and optimized. Primary mixed glial cell cultures were generated from heterozygous CX3CR-1+/EGFP mice, electrically stimulated for 4h/day over 3days using 75μm platinum-iridium microelectrodes, and biomarker immunofluorescence was measured. Electrodes were then imaged on a scanning electron microscope to assess sustained electrode damage. Fluorescence and electron microscopy analyses suggest varying degrees of localized responses for each biomarker assayed (Hoescht, EGFP, GFAP, and IL-1β), a result that expands on comparable in vivo models. This system allows for the comparison of a breadth of electrical stimulation parameters, and opens another avenue through which neural interfacing device developers can improve biocompatibility and longevity of electrodes in tissue.

Serum Amyloid A3 Fuels a Feed-Forward Inflammatory Response to the Bacterial Amyloid Curli in the Enteric Nervous System

BackgroundMounting evidence suggests a role for the gastro-intestinal microbiome as a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastro-intestinal tract and its enteric nervous system. MethodsTo examine the early response, we challenged primary murine myenteric networks with curli, the prototypic bacterial amyloid, and performed shotgun RNA sequencing and multiplex ELISA. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. ResultsCurli induced a pro-inflammatory response, with marked upregulation of Serum Amyloid A3 (Saa3) and the secretion of several cytokines. This pro-inflammatory state was primarily induced in enteric glia, was accompanied by elevated levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant SAA3 was sufficient to recapitulate this specific pro-inflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Like curli, recombinant SAA3 caused a strong upregulation of Saa3 transcripts, indicating a feed-forward loop. Colonization of curli-producing Salmonella and Dextran Sulphate Sodium (DSS)-induced colitis caused a significant increase in Saa3 transcripts, indicating a central role for SAA3 in enteric dysfunction. Inhibition of dual leucine zipper kinase (DLK), an upstream regulator of the c-Jun N-terminal kinase (JNK) pathway responsible for SAA3 production, attenuated curli- and SAA3-induced Saa3 upregulation, DNA damage and replication in enteric glia. ConclusionsOur results position SAA3 as an important mediator of gastro-intestinal vulnerability towards bacterial-derived amyloids and demonstrate the potential of DLK inhibition to dampen enteric pathology.

Physics-driven universal twin-image removal network for digital in-line holographic microscopy.

Digital in-line holographic microscopy (DIHM) enables efficient and cost-effective computational quantitative phase imaging with a large field of view, making it valuable for studying cell motility, migration, and bio-microfluidics. However, the quality of DIHM reconstructions is compromised by twin-image noise, posing a significant challenge. Conventional methods for mitigating this noise involve complex hardware setups or time-consuming algorithms with often limited effectiveness. In this work, we propose UTIRnet, a deep learning solution for fast, robust, and universally applicable twin-image suppression, trained exclusively on numerically generated datasets. The availability of open-source UTIRnet codes facilitates its implementation in various DIHM systems without the need for extensive experimental training data. Notably, our network ensures the consistency of reconstruction results with input holograms, imparting a physics-based foundation and enhancing reliability compared to conventional deep learning approaches. Experimental verification was conducted among others on live neural glial cell culture migration sensing, which is crucial for neurodegenerative disease research.

In vitro investigation of the effects of high-intensity therapeutic ultrasound (HITU) in glial tumor cell culture.

Amidst the evident challenges posed by brain tumors and the evident limitations of conventional treatment methodologies like surgery, radiotherapy, and chemotherapy, our primary objective was to probe the therapeutic potential of high-intensity therapeutic ultrasound (HITU). The aim was to introduce a safer, cost-effective, and efficient alternative to existing treatments, especially beneficial for inaccessible brain tumor sites and resource-constrained medical facilities. Leveraging post-1990s MR technology advancements, we employed the non-invasive HITU technique, akin to high-intensity focused ultrasound. This method directs acoustic energy to tissues, primarily inducing coagulation necrosis by absorbing energy and elevating tissue temperatures. Glial tumor cells were subjected to HITU to assess its effects. Upon applying HITU to glial tumor cells, significant alterations in cellular structural integrity were evident. The main action of HITU was the absorption of acoustic energy, leading to a notable temperature rise and coagulation necrosis. Flow cytometry indicated significant cellular changes post-HITU. ANOVA and t-test analyses showed a significant relationship between HITU application and time (p<0.05). The Shapiro-Wilk test revealed non-normal data distribution (p<0.05), leading to the use of nonparametric methods. The t-test results after HITU displayed significant differences (p<0.05) in cell counts and fluorescence intensity between control and treated groups. This result was consistent across multiple tests, indicating the reliability of the method in causing cellular damage to the tumor cells. Our laboratory analyses offer compelling evidence that HITU is not merely feasible but is also a promising non-invasive approach in the treatment paradigm of brain tumors. Standing distinctively apart from radiotherapy, HITU averts early, or late complications commonly associated with the former. While the path ahead mandates comprehensive research to ascertain its clinical utility, preliminary indications firmly posit HITU as a groundbreaking prospect in the management of brain tumors.

Protective effects of pyrroloquinoline quinone in brain folate deficiency

BackgroundFolates (Vitamin B9) are critical for normal neurodevelopment and function, with transport mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Cerebral folate uptake primarily occurs at the blood-cerebrospinal fluid barrier (BCSFB) through concerted actions of FRα and PCFT, with impaired folate transport resulting in the neurological disorder cerebral folate deficiency (CFD). Increasing evidence suggests that disorders associated with CFD also present with neuroinflammation, oxidative stress, and mitochondrial dysfunction, however the role of brain folate deficiency in inducing these abnormalities is not well-understood. Our laboratory has identified the upregulation of RFC by nuclear respiratory factor 1 (NRF-1) at the blood–brain barrier (BBB) once indirectly activated by the natural compound pyrroloquinoline quinone (PQQ). PQQ is also of interest due to its anti-inflammatory, antioxidant, and mitochondrial biogenesis effects. In this study, we examined the effects of folate deficiency and PQQ treatment on inflammatory and oxidative stress responses, and changes in mitochondrial function.MethodsPrimary cultures of mouse mixed glial cells exposed to folate-deficient (FD) conditions and treated with PQQ were analyzed for changes in gene expression of the folate transporters, inflammatory markers, oxidative stress markers, and mitochondrial DNA (mtDNA) content through qPCR analysis. Changes in cellular reactive oxygen species (ROS) levels were analyzed in vitro through a DCFDA assay. Wildtype (C57BL6/N) mice exposed to FD (0 mg/kg folate), or control (2 mg/kg folate) diets underwent a 10-day (20 mg/kg/day) PQQ treatment regimen and brain tissues were collected and analyzed.ResultsFolate deficiency resulted in increased expression of inflammatory and oxidative stress markers in vitro and in vivo, with increased cellular ROS levels observed in mixed glial cells as well as a reduction of mitochondrial DNA (mtDNA) content observed in FD mixed glial cells. PQQ treatment was able to reverse these changes, while increasing RFC expression through activation of the PGC-1α/NRF-1 signaling pathway.ConclusionThese results demonstrate the effects of brain folate deficiency, which may contribute to the neurological deficits commonly seen in disorders of CFD. PQQ may represent a novel treatment strategy for disorders associated with CFD, as it can increase folate uptake, while in parallel reversing many abnormalities that arise with brain folate deficiency.

β-adrenergic signaling triggers enteric glial reactivity and acute enteric gliosis during surgery

BackgroundEnteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma.MethodsSox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of β-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI.ResultsWith ~ 4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of β-adrenergic downstream signaling triggered enteric glial reactivity. Finally, distinct adrenergic agonists revealed β-1/2 adrenoceptors as the molecular targets of sympathetic–driven enteric glial reactivity.ConclusionsEnteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active β-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

Iguratimod Ameliorates the Severity of Secondary Progressive Multiple Sclerosis in Model Mice by Directly Inhibiting IL-6 Production and Th17 Cell Migration via Mitigation of Glial Inflammation.

We previously reported a novel secondary progressive multiple sclerosis (SPMS) model, progressive experimental autoimmune encephalomyelitis (pEAE), in oligodendroglia-specific Cx47-inducible conditional knockout (Cx47 icKO) mice. Based on our prior study showing the efficacy of iguratimod (IGU), an antirheumatic drug, for acute EAE treatment, we aimed to elucidate the effect of IGU on the SPMS animal model. We induced pEAE by immunizing Cx47 icKO mice with myelin oligodendrocyte glycoprotein peptide 35-55. IGU was orally administered from 17 to 50 days post-immunization. We also prepared a primary mixed glial cell culture and measured cytokine levels in the culture supernatant after stimulation with designated cytokines (IL-1α, C1q, TNF-α) and lipopolysaccharide. A migration assay was performed to evaluate the effect of IGU on the migration ability of T cells toward mixed glial cell cultures. IGU treatment ameliorated the clinical signs of pEAE, decreased the demyelinated area, and attenuated glial inflammation on immunohistochemical analysis. Additionally, IGU decreased the intrathecal IL-6 level and infiltrating Th17 cells. The migration assay revealed reduced Th17 cell migration and IL-6 levels in the culture supernatant after IGU treatment. Collectively, IGU successfully mitigated the clinical signs of pEAE by suppressing Th17 migration through inhibition of IL-6 production by proinflammatory-activated glial cells.

Influenza A virus infection disrupts oligodendrocyte homeostasis and alters the myelin lipidome in the adult mouse

BackgroundRecent data suggest that myelin may be altered by physiological events occurring outside of the central nervous system, which may cause changes to cognition and behavior. Similarly, peripheral infection by non-neurotropic viruses is also known to evoke changes to cognition and behavior.MethodsMice were inoculated with saline or influenza A virus. Bulk RNA-seq, lipidomics, RT-qPCR, flow cytometry, immunostaining, and western blots were used to determine the effect of infection on OL viability, protein expression and changes to the lipidome. To determine if microglia mediated infection-induced changes to OL homeostasis, mice were treated with GW2580, an inhibitor of microglia activation. Additionally, conditioned medium experiments using primary glial cell cultures were also used to test whether secreted factors from microglia could suppress OL gene expression.ResultsTranscriptomic and RT-qPCR analyses revealed temporal downregulation of OL-specific transcripts with concurrent upregulation of markers characteristic of cellular stress. OLs isolated from infected mice had reduced cellular expression of myelin proteins compared with those from saline-inoculated controls. In contrast, the expression of these proteins within myelin was not different between groups. Similarly, histological and immunoblotting analysis performed on various brain regions indicated that infection did not alter OL viability, but increased expression of a cellular stress marker. Shot-gun lipidomic analysis revealed that infection altered the lipid profile within the prefrontal cortex as well as in purified brain myelin and that these changes persisted after recovery from infection. Treatment with GW2580 during infection suppressed the expression of genes associated with glial activation and partially restored OL-specific transcripts to baseline levels. Finally, conditioned medium from activated microglia reduced OL-gene expression in primary OLs without altering their viability.ConclusionsThese findings show that peripheral respiratory viral infection with IAV is capable of altering OL homeostasis and indicate that microglia activation is likely involved in the process.

The S1P receptor 1 antagonist Ponesimod reduces TLR4-induced neuroinflammation and increases Aβ clearance in 5XFAD mice.

Previously, we showed that the sphingosine-1-phosphate (S1P) transporter spinster 2 (Spns2) mediates activation of microglia in response to amyloid β peptide (Aβ). Here, we investigated if Ponesimod, a functional S1P receptor 1 (S1PR1) antagonist, prevents Aβ-induced activation of glial cells and Alzheimer's disease (AD) pathology. We used primary cultures of glial cells and the 5XFAD mouse model to determine the effect of Aβ and Ponesimod on glial activation, Aβ phagocytosis, cytokine levels and pro-inflammatory signaling pathways, AD pathology, and cognitive performance. Aβ42 increased the levels of TLR4 and S1PR1, leading to their complex formation. Ponesimod prevented the increase in TLR4 and S1PR1 levels, as well as the formation of their complex. It also reduced the activation of the pro-inflammatory Stat1 and p38 MAPK signaling pathways, while activating the anti-inflammatory Stat6 pathway. This was consistent with increased phagocytosis of Aβ42 in primary cultured microglia. In 5XFAD mice, Ponesimod decreased the levels of TNF-α and CXCL10, which activate TLR4 and Stat1. It also increased the level of IL-33, an anti-inflammatory cytokine that promotes Aβ42 phagocytosis by microglia. As a result of these changes, Ponesimod decreased the number of Iba-1+ microglia and GFAP+astrocytes, and the size and number of amyloid plaques, while improving spatial memory as measured in a Y-maze test. Ponesimod targeting S1PR1 is a promising therapeutic approach to reprogram microglia, reduce neuroinflammation, and increase Aβ clearance in AD. NIHR01AG064234, RF1AG078338, R21AG078601, VAI01BX003643.