Glial Cell Cultures Research Articles

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Dendrimer-enabled targeted delivery attenuates glutamate excitotoxicity and improves motor function in a rabbit model of cerebral palsy.

Glutamate carboxypeptidase II (GCPII), localized on the surface of astrocytes and activated microglia, regulates extracellular glutamate concentration in the central nervous system (CNS). We have previously shown that GCPII is upregulated in activated microglia in the presence of inflammation. Inhibition of GCPII activity could reduce glutamate excitotoxicity, which may decrease inflammation and promote a 'normal' microglial phenotype. 2-(3-Mercaptopropyl) pentanedioic acid (2-MPPA) is the first GCPII inhibitor that underwent clinical trials. Unfortunately, immunological toxicities have hindered 2-MPPA clinical translation. Targeted delivery of 2-MPPA specifically to activated microglia and astrocytes that over-express GCPII has the potential to mitigate glutamate excitotoxicity and attenuate neuroinflammation. In this study, we demonstrate that 2-MPPA when conjugated to generation-4, hydroxyl-terminated polyamidoamine (PAMAM) dendrimers (D-2MPPA) localize specifically in activated microglia and astrocytes only in newborn rabbits with cerebral palsy (CP), not in controls. D-2MPPA treatment led to higher 2-MPPA levels in the injured brain regions compared to 2-MPPA treatment, and the extent of D-2MPPA uptake correlated with the injury severity. D-2MPPA was more efficacious than 2-MPPA in decreasing extracellular glutamate level in ex vivo brain slices of CP kits, and in increasing transforming growth factor beta 1 (TGF-β1) level in primary mixed glial cell cultures. A single systemic intravenous dose of D-2MPPA on postnatal day 1 (PND1) decreased microglial activation and resulted in a change in microglial morphology to a more ramified form along with amelioration of motor deficits by PND5. These results indicate that targeted dendrimer-based delivery specifically to activated microglia and astrocytes can improve the efficacy of 2-MPPA by attenuating glutamate excitotoxicity and microglial activation.

Drug repositioning targeting glutaminase reveals drug candidates for the treatment of Alzheimer’s disease patients

BackgroundDespite numerous clinical trials and decades of endeavour, there is still no effective cure for Alzheimer's disease. Computational drug repositioning approaches may be employed for the development of new treatment strategies for Alzheimer’s patients since an extensive amount of omics data has been generated during pre-clinical and clinical studies. However, targeting the most critical pathophysiological mechanisms and determining drugs with proper pharmacodynamics and good efficacy are equally crucial in drug repurposing and often imbalanced in Alzheimer’s studies.MethodsHere, we investigated central co-expressed genes upregulated in Alzheimer’s disease to determine a proper therapeutic target. We backed our reasoning by checking the target gene’s estimated non-essentiality for survival in multiple human tissues. We screened transcriptome profiles of various human cell lines perturbed by drug induction (for 6798 compounds) and gene knockout using data available in the Connectivity Map database. Then, we applied a profile-based drug repositioning approach to discover drugs targeting the target gene based on the correlations between these transcriptome profiles. We evaluated the bioavailability, functional enrichment profiles and drug-protein interactions of these repurposed agents and evidenced their cellular viability and efficacy in glial cell culture by experimental assays and Western blotting. Finally, we evaluated their pharmacokinetics to anticipate to which degree their efficacy can be improved.ResultsWe identified glutaminase as a promising drug target. Glutaminase overexpression may fuel the glutamate excitotoxicity in neurons, leading to mitochondrial dysfunction and other neurodegeneration hallmark processes. The computational drug repurposing revealed eight drugs: mitoxantrone, bortezomib, parbendazole, crizotinib, withaferin-a, SA-25547 and two unstudied compounds. We demonstrated that the proposed drugs could effectively suppress glutaminase and reduce glutamate production in the diseased brain through multiple neurodegeneration-associated mechanisms, including cytoskeleton and proteostasis. We also estimated the human blood–brain barrier permeability of parbendazole and SA-25547 using the SwissADME tool.ConclusionsThis study method effectively identified an Alzheimer’s disease marker and compounds targeting the marker and interconnected biological processes by use of multiple computational approaches. Our results highlight the importance of synaptic glutamate signalling in Alzheimer’s disease progression. We suggest repurposable drugs (like parbendazole) with well-evidenced activities that we linked to glutamate synthesis hereby and novel molecules (SA-25547) with estimated mechanisms for the treatment of Alzheimer’s patients.

Preparation of Primary Mixed Glial Cell Cultures from Adult Mouse Spinal Cord Tissue.

Central nervous system glial cells are known to mediate many neurocognitive/neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Similar glial responses have been recognized as critical factors contributing to the development of diseases in the peripheral nervous system, including various types of peripheral neuropathies, such as peripheral nerve injury-induced neuropathic pain, diabetic neuropathy, and HIV-associated sensory neuropathy. Investigation of the central mechanisms of these peripherally-manifested diseases often requires the examination of spinal cord glial cells at cellular/molecular levels in vitro. When using rodent models to study these diseases, many investigators have chosen to use neonatal cerebral cortices to prepare glial cultures or immortalized cell lines in order to obtain sufficient numbers of cells for assessment. However, differences in responses between cell lines versus primary cultures, neonatal vs. adult cells, and brain vs. spinal cord cells may result in misleading data. Here, we describe a protocol for preparing mixed glial cells from adult mouse spinal cord that can be used for direct in vitro evaluations or further preparation of microglia-enriched and microglia-depleted cells. In this protocol, spinal cord tissue is enzymatically dissociated and adult mixed glial cells are ready to be used between 12 and 14 days after the establishment of the culture. This protocol may be further refined to prepare spinal cord glial cells from spinal cord tissues of adult rats and potentially other species. Mixed glial cultures can be prepared from animals of different strains or post-in vivo manipulations and therefore are suitable for studying a variety of diseases/disorders that involve spinal cord pathological changes, such as amyotrophic lateral sclerosis and multiple sclerosis, as well as toxin-induced changes. © 2023 Wiley Periodicals LLC. Basic Protocol: Preparation of primary mixed glial cell cultures from adult mouse spinal cord tissue.

Role of interleukin-6 and interleukin-10 in morphological and functional changes of the blood–brain barrier in hypertriglyceridemia

BackgroundHypertriglyceridemia is closely linked to atherosclerosis related inflammatory processes and blood–brain barrier (BBB) dysfunction. Using apolipoprotein B-100 (APOB-100) transgenic mice, an animal model of chronic hypertriglyceridemia, we analyzed BBB function and morphology in vitro and ex vivo. Our objective was to determine which BBB characteristics are produced mainly by interleukin (IL)-6, an atherosclerosis promoting cytokine, and whether these actions can be antagonized by IL-10, an anti-inflammatory cytokine.MethodsBrain endothelial and glial cell cultures and brain microvessels were isolated from wild type (WT) and APOB-100 transgenic mice and were treated with IL-6, IL-10 and their combination. First, IL-6 and IL-10 production was measured in WT and APOB-100 microvessels using qPCR. Then functional parameters of endothelial cell cultures were analyzed and immunocytochemistry for key BBB proteins was performed.ResultsIL-6 mRNA levels were higher in brain microvessels than in brain parenchyma of APOB-100 transgenic mice. Transendothelial electric resistance and P-glycoprotein activity were lower, and paracellular permeability was higher in cultured APOB-100 brain endothelial cells. These features were sensitive to both IL-6 and IL-10 treatments. A decreased P-glycoprotein immunostaining was measured in transgenic endothelial cells under control conditions and in WT cells after treating them with IL-6. This effect was antagonized by IL-10. Changes in immunostaining for tight junction proteins were observed after IL-6 exposure, which were in part antagonized by IL-10. In glial cell cultures an increase in aquaporin-4 immunolabeling in the transgenic group and an increase in microglia cell density in WT glia cultures was detected after IL-6 treatment, which was antagonized by IL-10. In isolated brain microvessels a decrease in P-glycoprotein immunolabeled area fraction was measured in APOB-100 microvessels under control conditions and in WT microvessels after every cytokine treatment. ZO-1 immunolabeling showed characteristics similar to that of P-glycoprotein. No change was seen in claudin-5 and occludin immunoreactive area fractions in microvessels. A decrease in aquaporin-4 immunoreactivity was measured in WT microvessels treated by IL-6, which was antagonized by IL-10.ConclusionIL-6 produced in microvessels contributes to BBB impairment observed in the APOB-100 mice. We showed that IL-10 partly antagonizes the effects of IL-6 at the BBB.

A multiplexed siRNA screen identifies key kinase signaling networks of brain glia.

The dynamic behaviors of brain glial cells in various neuroinflammatory conditions and neurological disorders have been reported; however, little is known about the underlying intracellular signaling pathways. Here, we developed a multiplexed kinome-wide siRNA screen to identify the kinases regulating several inflammatory phenotypes of mouse glial cells in culture, including inflammatory activation, migration, and phagocytosis of glia. Subsequent proof-of-concept experiments involving genetic and pharmacological inhibitions indicated the importance of T-cell receptor signaling components in microglial activation and a metabolic shift from glycolysis to oxidative phosphorylation in astrocyte migration. This time- and cost-effective multiplexed kinome siRNA screen efficiently provides exploitable drug targets and novel insight into the mechanisms underlying the phenotypic regulation of glial cells and neuroinflammation. Moreover, the kinases identified in this screen may be relevant in other inflammatory diseases and cancer, wherein kinases play a critical role in disease signaling pathways.

Glial Cultures Differentiated from iPSCs of Patients with PARK2-Associated Parkinson's Disease Demonstrate a Pro-Inflammatory Shift and Reduced Response to TNFα Stimulation.

Parkinson's disease (PD) is the second most common neurodegenerative diseases characterized by progressive loss of midbrain dopaminergic neurons in the substantia nigra. Mutations in the PARK2 gene are a frequent cause of familial forms of PD. Sustained chronic neuroinflammation in the central nervous system makes a significant contribution to neurodegeneration events. In response to inflammatory factors produced by activated microglia, astrocytes change their transcriptional programs and secretion profiles, thus acting as immunocompetent cells. Here, we investigated iPSC-derived glial cell cultures obtained from healthy donors (HD) and from PD patients with PARK2 mutations in resting state and upon stimulation by TNFα. The non-stimulated glia of PD patients demonstrated higher IL1B and IL6 expression levels and increased IL6 protein synthesis, while BDNF and GDNF expression was down-regulated when compared to that of the glial cells of HDs. In the presence of TNFα, all of the glial cultures displayed a multiplied expression of genes encoding inflammatory cytokines: TNFA, IL1B, and IL6, as well as IL6 protein synthesis, although PD glia responded to TNFα stimulation less strongly than HD glia. Our results demonstrated a pro-inflammatory shift, a suppression of the neuroprotective gene program, and some depletion of reactivity to TNFα in PARK2-deficient glia compared to glial cells of HDs.

Identification of a Novel Dual Inhibitor of Acetylcholinesterase and Butyrylcholinesterase: In Vitro and In Silico Studies.

The enhancement of cholinergic functions via acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition is considered a valuable therapeutic strategy for the treatment of Alzheimer's disease. This study aimed to evaluate the in vitro effect of ZINC390718, previously filtered using computational approaches, on both cholinesterases and to characterize, using a molecular dynamics (MD) simulation, the possible binding mode of this compound inside the cholinesterase enzymes. The in vitro cytotoxicity effect was also investigated using a primary astrocyte-enriched glial cell culture. ZINC390718 presented in vitro dual inhibitory activity against AChE at a high micromolar range (IC50 = 543.8 µM) and against BuChE (IC50 = 241.1 µM) in a concentration-dependent manner, with greater activity against BuChE. The MD simulation revealed that ZINC390718 performed important hydrophobic and H-bond interactions with the catalytic residue sites on both targets. The residues that promoted the hydrophobic interactions and H-bonding in the AChE target were Leu67, Trp86, Phe123, Tyr124, Ser293, Phe295, and Tyr341, and on the BuChE target, they were Asp70, Tyr332, Tyr128, Ile442, Trp82, and Glu197. The cytotoxic effect of Z390718, evaluated via cell viability, showed that the molecule has low in vitro toxicity. The in vitro and in silico results indicate that ZINC390718 can be used as chemotype for the optimization and identification of new dual cholinesterase inhibitors.

Dissociation and Culture of Adult Mouse Satellite Glial Cells.

Satellite glial cells (SGCs) are a type of glial cell population that originates from neural crest cells. They ultimately migrate to surround the cell bodies of neurons in the ganglia of the peripheral nervous system. Under physiological conditions, SGCs perform homeostatic functions by modifying the microenvironment around nearby neurons and provide nutrients, structure, and protection. In recent years, they have gained considerable attention due to their involvement in peripheral nerve regeneration and pain. Although methods for culturing neonatal or rat SGCs have long existed, a well-characterized method for dissociating and culturing adult SGCs from mouse tissues has been lacking until recently. This has impeded further studies of their function and the testing of new therapeutics. This protocol provides a detailed description of how to obtain primary cultures of adult SGCs from mouse dorsal root ganglia in approximately two weeks with over 90% cell purity. We also demonstrate cell purity of these cultures using quantitative real-time RT-PCR and their functional integrity using calcium imaging. Key features • Detailed and simplified protocol to dissociate and culture primary satellite glial cells (SGCs) from adult mice. • Cells are dissociated in approximately 2-3 h and cultured for approximately two weeks. • These SGC cultures allow both molecular and functional studies.

Uterine contractility in aged rats after administration of conditioned media from cryopreserved glial cell culture

Uterine contractility in aged rats after administration of conditioned media from cryopreserved glial cell culture

Chemical signaling in the developing avian retina: Focus on cyclic AMP and AKT-dependent pathways.

Communication between developing progenitor cells as well as differentiated neurons and glial cells in the nervous system is made through direct cell contacts and chemical signaling mediated by different molecules. Several of these substances are synthesized and released by developing cells and play roles since early stages of Central Nervous System development. The chicken retina is a very suitable model for neurochemical studies, including the study of regulation of signaling pathways during development. Among advantages of the model are its very well-known histogenesis, the presence of most neurotransmitter systems found in the brain and the possibility to make cultures of neurons and/or glial cells where many neurochemical functions develop in a similar way than in the intact embryonic tissue. In the chicken retina, some neurotransmitters or neuromodulators as dopamine, adenosine, and others are coupled to cyclic AMP production or adenylyl cyclase inhibition since early stages of development. Other substances as vitamin C and nitric oxide are linked to the major neurotransmitter glutamate and AKT metabolism. All these different systems regulate signaling pathways, including PKA, PKG, SRC, AKT and ERK, and the activation of the transcription factor CREB. Dopamine and adenosine stimulate cAMP accumulation in the chick embryo retina through activation of D1 and A2a receptors, respectively, but the onset of dopamine stimulation is much earlier than that of adenosine. However, adenosine can inhibit adenylyl cyclase and modulate dopamine-dependent cAMP increase since early developmental stages through A1 receptors. Dopamine stimulates different PKA as well as EPAC downstream pathways both in intact tissue and in culture as the CSK-SRC pathway modulating glutamate NMDA receptors as well as vitamin C release and CREB phosphorylation. By the other hand, glutamate modulates nitric oxide production and AKT activation in cultured retinal cells and this pathway controls neuronal survival in retina. Glutamate and adenosine stimulate the release of vitamin C and this vitamin regulates the transport of glutamate, activation of NMDA receptors and AKT phosphorylation in cultured retinal cells. In the present review we will focus on these reciprocal interactions between neurotransmitters or neuromodulators and different signaling pathways during retinal development.

Cathelicidin-Related Antimicrobial Peptide Negatively Regulates Bacterial Endotoxin-Induced Glial Activation

Recent studies have suggested that mouse cathelicidin-related antimicrobial peptide (CRAMP) and its human homologue leucine leucine-37 (LL-37) play critical roles in innate immune responses. Here, we studied the role of mouse CRAMP in bacterial endotoxin lipopolysaccharide (LPS)-induced neuroinflammation. CRAMP peptide treatment significantly inhibited LPS-mediated inflammatory activation of glial cells in culture. In the animal model of LPS-induced neuroinflammation, CRAMP expression was highly induced in multiple cell types, such as astrocytes, microglia, and neurons. Injection of exogenous CRAMP peptide significantly inhibited inflammatory cytokine expression and the reactivity of glial cells in the mouse brain following intraperitoneal or intracerebroventricular LPS administration. Altogether, results of the study suggest that CRAMP plays an important part in containment of LPS-induced neuroinflammatory responses, and that CRAMP can be exploited for the development of targeted therapies for neuroinflammatory conditions associated with bacterial infection.

Neuroprotective action of α-Klotho against LPS-activated glia conditioned medium in primary neuronal culture

The α-Klotho is an anti-aging protein that, when overexpressed, extends the life span in humans and mice. It has an anti-inflammatory and protective action on renal cells by inhibiting NF-κB activation and production of inflammatory cytokines in response to TNF-α. Furthermore, studies have shown the neuroprotective effect of α-Klotho against neuroinflammation on different conditions, such as aging, animal models of neurodegenerative diseases, and ischemic brain injury. This work aimed to evaluate the effects of α-Klotho protein on primary glial cell culture against the proinflammatory challenge with LPS and how this could interfere with neuronal health. Cortical mixed glial cells and purified astrocytes were pretreated with α- α-Klotho and stimulated with LPS followed by TNFα, IL-1β, IL-6, IFN-γ levels, and NF-κB activity analysis. Conditioned medium from cortical mixed glia culture treated with LPS (glia conditioned medium (GCM) was used to induce neuronal death of primary cortical neuronal culture and evaluate if GCM-KL (medium from glia culture pretreated α-Klotho followed by LPS stimulation) or GCM + LPS in the presence of KL can reverse the effect. LPS treatment in glial cells induced an increase in proinflammatory mediators such as TNF-α, IL-1β, IL-6, and IFN-γ, and activation of astrocyte NF-κB. GCM treated-cortical neuronal culture induced a concentration-dependent neuronal death. Pretreatment with α-Klotho decreased TNF-α and IL-6 production, reverted NF-κB activation, and decreased neuronal death induced by GCM. In addition, KL incubation together with GCM + LPS completely reverts the neuronal toxicity induced by low concentration of GCM-LPS. These data suggest an anti-inflammatory and neuroprotective effect of α-Klotho protein in the CNS. This work demonstrated the therapeutic potential of α-Klotho in pathological processes which involves a neuroinflammatory component.

Isoliquiritigenin Protects Neuronal Cells against Glutamate Excitotoxicity.

It is considered that glutamate excitotoxicity may be a major factor in the pathological death of neurons and mediate the development of neurodegenerative diseases in humans. Here, we show that isoliquiritigenin (ILG) at a concentration of 0.5-5 µM protects primary neuroglial cell culture from glutamate-induced death (glutamate 100 µM). ILG (1 µM) prevented a sharp increase in [Ca2+]i and a decrease in mitochondrial potential (ΔΨm). With the background action of ILG (1-5 µM), there was an increase in oxygen consumption rate (OCR) in response to glutamate, as well as in reserve respiration. The neuroprotective effect of ILG (5 µM) was accompanied by an increase in non-mitochondrial respiration. The results show that ILG can protect cortical neurons from death by preventing the development of calcium deregulation and limiting mitochondrial dysfunction caused by a high dose of glutamate. We hypothesize that ILG will be useful in drug development for the prevention or treatment of neurodegenerative diseases accompanied by glutamate excitotoxicity.

Anti-inflammatory Action of the Treated-Yeast, Milmed, Under IBS-IBD Conditions

In order to assess the anti-inflammatory and therapeutic action of the probiotic, treated-yeast Milmed, twelve patients (age range 25-to-79 years) presenting IBS-IBD with a wide range of gut-intestinal symptoms, were studied. Each patient completed a questionnaire outlining demographic characteristics and test items regarding their health issues, both prior to and after the twelve-week period (on three occasions each week) of Milmed intervention. Patients’ accounts of their symptom-profiles were quantified and subjected to statistical analyses. It was observed that in comparison with the placebo (administered untreated-yeast) control, the Milmed group reported fewer symptoms, following treatment, as well as also fewer symptoms compared to their pre-treatment report. Untreated-yeast administration to patients did not induce any reduction of IBS-IBD symptoms. There was no correlation between patients’ responses regarding symptoms Before and After intervention. It is possible that increasing the number of capsules ingested per week, from 3/week to 1/day, may have provided the patients with greater benefits. Despite certain limitations of this study when taken together with the reported anti-inflammatory propensity of Milmed upon glial and neuronal cell cultures in vitro, these findings imply several useful therapeutic applications for the treated-yeast, Milmed, in the treatment of gut-intestinal conditions, such as IBS-IBD and other related ailments.

Analog of kynurenic acid decreases tau pathology by modulating astrogliosis in rat model for tauopathy.

Kynurenines have immunomodulatory and neuroactive properties and can influence the central nervous system. Previous studies showed the involvement of the kynurenines in the pathogenesis and progression of neurodegenerative disease. In neurodegenerative disorders, including tauopathies, the tryptophan metabolism is shifted toward neurotoxic agents and the reduction of neuroprotectant products. Astrocyte-derived kynurenic acid serves as a neuroprotectant. However, systemic administration of kynurenic acid is not effective because of low permeability across the blood-brain barrier (BBB). We used a kynurenic acid analog with similar biological activity but higher brain permeability to overcome BBB limitations. In the present study, we used amide derivate of kynurenic acid N-(2-N, N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamid (KYNA-1). We administered KYNA-1 for three months to tau transgenic rats SHR-24 and analyzed the effect on tau pathology and activation of glial cells. Primary glial cell cultures were applied to identify the mechanism of the KYNA-1 effect. KYNA-1 was not toxic to rats after chronic three-month administration. When chronically administered, KYNA-1 reduced hyperphosphorylation of insoluble tau in the brain of transgenic rats. Noteworthily, the plasma total tau was also reduced. We determined that the effect of KYNA-1 on tau pathology was induced through the modulation of glial activation. KYNA-1 inhibited LPS induced activation of astrocytes and induced transformation of microglia to M2 phenotype. We identified that the administration of KYNA-1 reduced tau hyperphosphorylation and neuroinflammation. KYNA-1 may serve as a promising treatment for tauopathies.

Gabapentinoids Suppress Lipopolysaccharide-Induced Interleukin-6 Production in Primary Cell Cultures of the Rat Spinal Dorsal Horn

Introduction: Gabapentin and pregabalin are drugs to treat neuropathic pain. Several studies highlighted effects on presynaptic terminals of nociceptors. Via binding to α₂δ subunits of voltage-gated calcium channels, gabapentinoids modulate the synaptic transmission of nociceptive information. However, recent studies revealed further properties of these substances. Treatment with gabapentin or pregabalin in animal models of neuropathic pain resulted not only in reduced symptoms of hyperalgesia but also in an attenuated activation of glial cells and decreased production of pro-inflammatory mediators in the spinal dorsal horn. Methods: In the present study, we aimed to investigate the impact of gabapentinoids on the inflammatory response of spinal dorsal horn cells, applying the established model of neuro-glial primary cell cultures of the superficial dorsal horn (SDH). We studied effects of gabapentin and pregabalin on lipopolysaccharide (LPS)-induced cytokine release (bioassays), expression of inflammatory marker genes (RT-qPCR), activation of transcription factors (immunocytochemistry), and Ca²⁺ responses of SDH neurons to stimulation with substance P and glutamate (Ca²⁺-imaging). Results: We detected an attenuated LPS-induced expression and release of interleukin-6 by SDH cultures in the presence of gabapentinoids. In addition, a significant main effect of drug treatment was observed for mRNA expression of microsomal prostaglandin E synthase 1 and the inhibitor of nuclear factor kappa B. Nuclear translocation of inflammatory transcription factors in glial cells was not significantly affected by gabapentinoid treatment. Moreover, both substances did not modulate neuronal responses upon stimulation with substance P or glutamate. Conclusion: Our results provide evidence for anti-inflammatory capacities of gabapentinoids on the acute inflammatory response of SDH primary cultures upon LPS stimulation. Such effects may contribute to the pain-relieving effects of gabapentinoids.

Effects of ethanol and deferoxamine on rat primary glial cell cultures, in regard with ischemia induced by traumatic spinal cord injury

Although they have been regarded, in the past, as passive support cells, many experimental data have shown that glial cells play a critical role in the development and functioning of the nervous system. Despite the advances that have been made in understanding astrocytes' role in the nervous system's development and function, our knowledge of their interactions with other cells is still limited, albeit neurons are dependent on the trophic support provided by astrocytes release. Materials and Methods. The use of the McCarthy and de Vellis methods for isolating glial cells has been regarded as an essential tool for studying their function. This study aims to evaluate the effects of ethanol and deferoxamine on primary rat glial cell cultures and try to explain, as far as possible, the relevance of such effects for patients with chronic alcoholism and traumatic spinal cord injuries. Discussion. Because glial cells are very important in the functioning of the central nervous system and experiments cannot be performed on human primary nerve cell cultures, we performed an experiment on glial cells harvested from the newborn rat, analyzing the dynamics of IL-6 and TNF alpha on models of suffering in spinal cord injury (hypoxia and thermally stress). Conclusion. Inhibition of TNF alpha synthesis was more important at 7 days posttraumatic in cells with prolonged ethanolic exposure, even if protein levels of IL-6 were elevated (under similar experimental conditions). Thus, we can say that long-term exposure to ethanol of nerve cells can ensure a favorable evolution of medical recovery (by increasing TNF alpha), even if the inflammatory process remains active (shown by elevated IL-6 values). Keywords: ethyl alcohol, deferoxamine, primary glial cells cultures, traumatic Spinal Cord Injury

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells. The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

Methylenedioxy Piperamide-Derived Compound D5 Regulates Inflammatory Cytokine Secretion in a Culture of Human Glial Cells.

Neuroinflammation is the cornerstone of most neuronal disorders, particularly neurodegenerative diseases. During the inflammatory process, various pro-inflammatory cytokines, chemokines, and enzymes—such as interleukin 1-β (IL1-β), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthases (iNOS), inhibitory kappa kinase (IKK), and inducible nitric oxide (NO)—are over-expressed in response to every stimulus. Methods: In the present study, we focused on the anti-neuroinflammatory efficacy of (2E,4E)-N,5-bis(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienamide, encoded D5. We investigated the efficacy of D5 on the upstream and downstream products of inflammatory pathways in CHME3 and SVG cell lines corresponding to human microglia and astrocytes, respectively, using various in silico, in vitro, and in situ techniques. Results: The results showed that D5 significantly reduced the level of pro-inflammatory cytokines by up-regulating PPAR-γ expression and suppressing IKK-β, iNOS, NO production, and NF-κB activation in inflamed astrocytes (SVG) and microglia (CHME3) after 24 h of incubation. The data demonstrated remarkably higher efficacy of D5 compared to ASA (Aspirin) in reducing NF-κB-dependent neuroinflammation. Conclusions: We observed that the functional-group alteration had an extreme influence on the levels of druggability and the immunomodulatory properties of two analogs of piperamide, D5, and D4 ((2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-(hydroxymethyl)phenyl)penta-2,4-dienamide)). The present study suggested D5 as a potential anti-neuroinflammatory agent for further in vitro, in vivo, and clinical investigations.