Astroglial Cultures Research Articles

Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes.

Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

Nerve growth factor receptor (Ngfr) induces neurogenic plasticity by suppressing reactive astroglial Lcn2/Slc22a17 signaling in Alzheimer’s disease

Neurogenesis, crucial for brain resilience, is reduced in Alzheimer’s disease (AD) that induces astroglial reactivity at the expense of the pro-neurogenic potential, and restoring neurogenesis could counteract neurodegenerative pathology. However, the molecular mechanisms promoting pro-neurogenic astroglial fate despite AD pathology are unknown. In this study, we used APP/PS1dE9 mouse model and induced Nerve growth factor receptor (Ngfr) expression in the hippocampus. Ngfr, which promotes neurogenic fate of astroglia during the amyloid pathology-induced neuroregeneration in zebrafish brain, stimulated proliferative and neurogenic outcomes. Histological analyses of the changes in proliferation and neurogenesis, single-cell transcriptomics, spatial proteomics, and functional knockdown studies showed that the induced expression of Ngfr reduced the reactive astrocyte marker Lipocalin-2 (Lcn2), which we found was sufficient to reduce neurogenesis in astroglia. Anti-neurogenic effects of Lcn2 was mediated by Slc22a17, blockage of which recapitulated the pro-neurogenicity by Ngfr. Long-term Ngfr expression reduced amyloid plaques and Tau phosphorylation. Postmortem human AD hippocampi and 3D human astroglial cultures showed elevated LCN2 levels correlate with reactive gliosis and reduced neurogenesis. Comparing transcriptional changes in mouse, zebrafish, and human AD brains for cell intrinsic differential gene expression and weighted gene co-expression networks revealed common altered downstream effectors of NGFR signaling, such as PFKP, which can enhance proliferation and neurogenesis in vitro when blocked. Our study suggests that the reactive non-neurogenic astroglia in AD can be coaxed to a pro-neurogenic fate and AD pathology can be alleviated with Ngfr. We suggest that enhancing pro-neurogenic astroglial fate may have therapeutic ramifications in AD.

Organoprotective Properties of Argon (Review)

The history of studying the organoprotective properties of argon (Ar) began in 1998 when a group of Russian researchers investigated the effect of hypoxic gas mixtures on mammalian organisms. Over several decades, evidence of the cardio-, neuro-, and nephroprotective effects of argon in various diseases and conditions in experimental models in vivo and in vitro have been accumulated. However, the lack of clinical studies to date has prompted us to carry out a systematic review analyzing the results of preclinical studies revealing organoprotective properties of argon, which could provide a rationale for its future clinical studies.The aimof this review is to describe the mechanisms of organoprotective properties of argon determined in preclinical studies.Material and methods. The search yielded 266 articles. The search algorithm was developed in accordance with the requirements and reporting guidelines for systematic reviews and meta-analysis (PRISMA) in the PubMed and Google Scholar databases. The methodology included using search queries, keywords (including MeSH), and logical operators. The keywords used for the search in the PubMed and Google Scholar databases were «argon», «ar», «protection», and «mechanism». The review included in vivo and in vitro studies.Results.The following mechanisms of argon action were identified: activation of N-terminal c-Jun kinase(JNK), p38(ERK1/2), and ERK1/2 in models of airway epithelial cells, neuronal and astroglial cell cultures, as well as in models of retinal ischemia and reperfusion injury in rats and a rabbit model of ischemia-reperfusion myocardium. Significant neuroprotective effects of argon and its influence on apoptosis were shown using small rodent models.Conclusion.The results of preclinical studies of argon have proved both its safety and organoprotective properties in in vitro and in vivo models. Analysis of the data provides a rationale for the initiation of clinical studies of argon, which could significantly improve outcomes in patients after cerebrovascular accidents, particularly post ischemic stroke.

Monitoring cell membrane recycling dynamics of proteins using whole-cell fluorescence recovery after photobleaching of pH-sensitive genetic tags.

Population behavior of signaling molecules on the cell surface is key to their adaptive function. Live imaging of proteins tagged with fluorescent molecules has been an essential tool in understanding this behavior. Typically, genetic or chemical tags are used to target molecules present throughout the cell, whereas antibody-based tags label the externally exposed molecular domains only. Both approaches could potentially overlook the intricate process of in-out membrane recycling in which target molecules appear or disappear on the cell surface. This limitation is overcome by using a pH-sensitive fluorescent tag, such as Super-Ecliptic pHluorin (SEP), because its emission depends on whether it resides inside or outside the cell. Here we focus on the main glial glutamate transporter GLT1 and describe a genetic design that equips GLT1 molecules with SEP without interfering with the transporter's main function. Expressing GLT1-SEP in astroglia in cultures or in hippocampal slices enables monitoring of the real-time dynamics of the cell-surface and cytosolic fractions of the transporter in living cells. Whole-cell fluorescence recovery after photobleaching and quantitative image-kinetic analysis of the resulting time-lapse images enables assessment of the rate of GLT1-SEP recycling on the cell surface, a fundamental trafficking parameter unattainable previously. The present protocol takes 15-20 d to set up cell preparations, and 2-3 d to carry out live cell experiments and data analyses. The protocol can be adapted to study different membrane molecules of interest, particularly those proteins whose lifetime on the cell surface is critical to their adaptive function.

FMRP-related retinal phenotypes: Evidence of glutamate-glutamine metabolic cycle impairment

FMRP, the fragile X mental retardation protein coded by the FMR1 gene, is an RNA-binding protein that assists transport, stabilization and translational regulation of specific synaptic mRNAs. Its expression has been found in multiple cell types of central nervous system (CNS) including glial cells where its involvement in glutamate neurotransmitter homeostasis have been shown. Indeed, glutamate homeostasis deficit has been observed in absence of FMRP in-vivo in cortex and hippocampus structures as well as in vitro on astroglial cell culture. Interestingly, the retina which is an extension of the CNS is presenting electrophysiological alterations in absence of FMRP in both human and murine models suggesting neurotransmitter impairments. Therefore, we investigate the consequences of Fmrp absence on Glutamate-Glutamine cycle in whole retinas and primary retinal Müller cells culture which are the main glial cells of the retina. Using the Fmr1-/y mice, we have shown in vivo and in vitro that the absence of Fmrp in Müller cells is characterized by loss of Glutamate-Glutamine cycle homeostasis due to a lower Glutamine Synthetase protein expression and activity. The lack of Fmrp in the retina induces a reduced flow of glutamine synthesis. Our data established for the first time in literature a direct link between the lack of Fmrp and neurotransmitter homeostasis in the retina.

Role of Macrophages and Mast Cells as Key Players in the Maintenance of Gastrointestinal Smooth Muscle Homeostasis and Disease.

The gut contains the largest macrophage pool in the body, with populations of macrophages residing in the mucosa and muscularis propria of the gastrointestinal (GI) tract. Muscularis macrophages (MMs), which are located within the muscularis propria, interact with cells essential for GI function, such as interstitial cells of Cajal, enteric neurons, smooth muscle cells, enteric glia, and fibroblast-like cells, suggesting that these immune cells contribute to several aspects of GI function. This review focuses on the latest insights on the factors contributing to MM heterogeneity and the functional interaction of MMs with other cell types essential for GI function. This review integrates the latest findings on macrophages in other organs with increasing knowledge of MMs to better understand their role in a healthy and diseased gut. We describe the factors that contribute to (muscularis macrophage) MM heterogeneity, and the nature of MM interactions with cells regulating GI function. Finally, we also describe the increasing evidence suggesting a critical role of another immune cell type, the mast cell, in normal and diseased GI physiology.

Comparison of the Effects of Chemokine Receptors CXCR2 and CXCR3 Pharmacological Modulation in Neuropathic Pain Model-In Vivo and In Vitro Study.

Recent findings have highlighted the roles of CXC chemokine family in the mechanisms of neuropathic pain. Our studies provide evidence that single/repeated intrathecal administration of CXCR2 (NVP-CXCR2-20) and CXCR3 ((±)-NBI-74330) antagonists explicitly attenuated mechanical/thermal hypersensitivity in rats after chronic constriction injury of the sciatic nerve. After repeated administration, both antagonists showed strong analgesic activity toward thermal hypersensitivity; however, (±)-NBI-74330 was more effective at reducing mechanical hypersensitivity. Interestingly, repeated intrathecal administration of both antagonists decreased the mRNA and/or protein levels of pronociceptive interleukins (i.e., IL-1beta, IL-6, IL-18) in the spinal cord, but only (±)-NBI-74330 decreased their levels in the dorsal root ganglia after nerve injury. Furthermore, only the CXCR3 antagonist influenced the spinal mRNA levels of antinociceptive factors (i.e., IL-1RA, IL-10). Additionally, antagonists effectively reduced the mRNA levels of pronociceptive chemokines; NVP-CXCR2-20 decreased the levels of CCL2, CCL6, CCL7, and CXCL4, while (±)-NBI-74330 reduced the levels of CCL3, CCL6, CXCL4, and CXCL9. Importantly, the results obtained from the primary microglial and astroglial cell cultures clearly suggest that both antagonists can directly affect the release of these ligands, mainly in microglia. Interestingly, NVP-CXCR2-20 induced analgesic effects after intraperitoneal administration. Our research revealed important roles for CXCR2 and CXCR3 in nociceptive transmission, especially in neuropathic pain.

SIRT-1 Activity Sustains Cholesterol Synthesis in the Brain

SIRT-1 is a potent energy regulator that has been implicated in the aging of different tissues, and cholesterol synthesis demands high amounts of cellular adenosine triphosphate. An efficient synaptic transmission depends on processes that are highly influenced by cholesterol levels, like endocytosis, exocytosis and membrane lateral diffusion of neurotransmitter receptors. We set out to investigate whether SIRT-1 activity affects brain cholesterol metabolism. We found that pharmacological inhibition of SIRT-1 with EX-527 reduces the mRNA amounts of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), Cytochrome P450 46A1 (CYP46A1) and Apolipoprotein E (APO-E) in rat primary cortical cultures. The decreased expression of these genes was paralleled by a significant reduction of the cholesterol levels in this type of neuronal culture. Interestingly, a cholesterol decrease of similar extent was observed in mouse astroglial cultures after EX-527 treatment. In agreement, mice administered with EX-527 for 5 days showed a down-regulation of cholesterol synthesis in the cortex, with significant reductions in the mRNA amounts of the transcription factor Sterol Regulatory Element Binding Protein 2 (SREBP-2) and the enzyme HMGCR, two key regulators of the cholesterol synthesis. These transcriptional changes were paralleled by reduced cholesterol levels at cortical synapses. SIRT-1 inhibition also reduced the amount of cholesterol in the hippocampus but without affecting the HMGCR expression levels. Altogether, these results uncover a role for SIRT-1 in the regulation of cholesterol metabolism, and demonstrate that SIRT-1 is required to sustain adequate levels of cholesterol synthesis in the adult brain.

Long-Lasting Changes in Glial Cells Isolated From Rats Subjected to the Valproic Acid Model of Autism Spectrum Disorder.

Synaptic alterations concomitant with neuroinflammation have been described in patients and experimental models of autism spectrum disorder (ASD). However, the role of microglia and astroglia in relation to synaptic changes is poorly understood. Male Wistar rats prenatally exposed to valproic acid (VPA, 450 mg/kg, i.p.) or saline (control) at embryonic day 10.5 were used to study synapses, microglia, and astroglia in the prefrontal cortex (PFC) at postnatal days 3 and 35 (PND3 and PND35). Primary cultures of cortical neurons, microglia, and astroglia isolated from control and VPA animals were used to study each cell type individually, neuron-microglia and microglia-astroglia crosstalk. In the PFC of VPA rats, synaptic changes characterized by an increase in the number of excitatory synapses were evidenced at PND3 and persisted until PND35. At PND3, microglia and astroglia from VPA animals were morphologically similar to those of age-matched controls, whereas at PND35, reactive microgliosis and astrogliosis were observed in the PFC of VPA animals. Cortical neurons isolated from VPA rats mimicked in vitro the synaptic pattern seen in vivo. Cortical microglia and astroglia isolated from VPA animals exhibited reactive morphology, increased pro-inflammatory cytokines, and a compromised miRNA processing machinery. Microglia from VPA animals also showed resistance to a phagocytic challenge. In the presence of neurons from VPA animals, microglia isolated from VPA rats revealed a non-reactive morphology and promoted neurite outgrowth, while microglia from control animals displayed a reactive profile and promoted dendritic retraction. In microglia-astroglia co-cultures, microglia from VPA animals displayed a reactive profile and exacerbated astrocyte reactivity. Our study indicates that cortical microglia from VPA animals are insensitive or adapted to neuronal cues expressed by neurons from VPA animals. Further, long-term in vivo microgliosis could be the result of altered microglia-astroglia crosstalk in VPA animals. Thus, our study highlights cortical microglia-astroglia communication as a new mechanism implicated in neuroinflammation in ASD; consequently, we propose that this crosstalk is a potential target for interventions in this disorder.

Contrasting Roles of Ang II and ACEA in the Regulation of IL10 and IL1β Gene Expression in Primary SHR Astroglial Cultures.

Angiotensin (Ang) II is well-known to have potent pro-oxidant and pro-inflammatory effects in the brain. Extensive crosstalk between the primary Ang II receptor, Ang type 1 receptor (AT1R), and the cannabinoid type 1 receptor (CB1R) has been demonstrated by various groups in the last decade. Since activation of glial CB1R has been demonstrated to play a key role in the resolution of inflammatory states, we investigated the role of Ang II (100 nM) and/or ACEA (10 nM), a potent CB1R-specific agonist in the regulation of inflammatory markers in astrocytes from spontaneously hypertensive rats (SHR) and Wistar rats. Astrocytes were cultured from brainstems and cerebellums of SHR and Wistar rats and assayed for IL1β and IL10 gene expression and secreted fraction, in treated and non-treated cells, by employing qPCR and ELISA, respectively. mRNA expression of both IL10 and IL1β were significantly elevated in untreated brainstem and cerebellar astrocytes isolated from SHR when compared to Wistar astrocytes. No changes were observed in the secreted fraction. While ACEA-treatment resulted in a significant increase in IL10 gene expression in Wistar brainstem astrocytes (Log2FC ≥ 1, p < 0.05), its effect in SHR brainstem astrocytes was diminished. Ang II treatment resulted in a strong inhibitory effect on IL10 gene expression in astrocytes from both brain regions of SHR and Wistar rats (Log2FC ≤ −1, p < 0.05), and an increase in IL1β gene expression in brainstem astrocytes from both strains (Log2FC ≥ 1, p < 0.05). Co-treatment of Ang II and ACEA resulted in neutralization of Ang II-mediated effect in Wistar brainstem and cerebellar astrocytes, but not SHR astrocytes. Neither Ang II nor ACEA resulted in any significant changes in IL10 or IL1β secreted proteins. These data suggest that Ang II and ACEA have opposing roles in the regulation of inflammatory gene signature in astrocytes isolated from SHR and Wistar rats. This however does not translate into changes in their secreted fractions.

Salidroside Inhibits Reactive Astrogliosis and Glial Scar Formation in Late Cerebral Ischemia via the Akt/GSK-3β Pathway.

Cerebral ischemia leads to reactive astrogliosis and glial scar formation. Glial scarring can impede functional restoration during the recovery phase of stroke. Salidroside has been shown to have neuroprotective effects after ischemic stroke, but its impact on long-term neurological recovery, especially whether it regulates reactive astrogliosis and glial scar formation, is unclear. In this study, male adult C57/BL6 mice were subjected to transient cerebral ischemia injury followed by intravenous salidroside treatment. Primary astrocytes were treated with lipopolysaccharide (LPS) or conditioned medium from cultured primary neurons subjected to oxygen-glucose deprivation (CM-OGD). Salidroside significantly improved long-term functional outcomes following ischemic stroke in the rotarod and corner tests. It also reduced brain glial scar volume and decreased expression of the glial scar marker, glial fibrillary acidic protein (GFAP) and inhibited astrocyte proliferation. In primary astrocyte cultures, salidroside protected astrocytes from CM-OGD injury-induced reactive astroglial proliferation, increasing the percentage of cells in G0/G1 phase and reducing the S populations. The inhibitory effect of salidroside on the cell cycle was related to downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4) mRNA expression and increased p27Kip1 mRNA expression. Similar results were found in the LPS-stimulated injury model in astroglial cultures. Western blot analysis demonstrated that salidroside attenuated the CM-OGD-induced upregulation of phosphorylated Akt and glycogen synthase kinase 3β (GSK-3β). Taken together, these results suggested that salidroside can inhibit reactive astrocyte proliferation, ameliorate glial scar formation and improve long-term recovery, probably through its effects on the Akt/GSK-3β pathway.

Kynurenic Acid Protects Against Reactive Glial-associated Reductions in the Complexity of Primary Cortical Neurons.

Brain glia produce neuroactive metabolites via tryptophan-kynurenine catabolism. A role for kynurenine pathway (KP) metabolites is proposed in reactive glial associated neurodegeneration. The aim of this investigation was to assess the role of KP induction and KP metabolites in driving reactive glial associated neuronal atrophy. Rat primary mixed glia, and enriched microglial and astroglial cultures were stimulated with IFNγ (10ng/ml) for 24 hours. KP induction in mixed glial cells was confirmed by raised expression of the rate limiting KP enzyme indoleamine 2,3 dioxygenase (IDO) and raised concentrations of KP metabolites kynurenic acid (KYNA) and quinolinic acid (QUIN) in the conditioned media. Conditioned media was transferred onto immature (3 days) and mature (21 days) primary cortical neurons in vitro for 24 hours. IFNγ-stimulated mixed glial conditioned media reduced neurite outgrowth and complexity of both immature and mature neurons and co-localised expression of synaptic markers determined by immunocytochemistry. Pre-treatment of mixed glial cells with the IDO inhibitor, 1-methyltryptophan (1-MT) (L) prevented these effects of IFNγ-stimulated mixed glial conditioned media. KYNA increased complexity and synapse formation in mature cortical neurons and protected against reduced neuronal complexity and co-localised expression of synaptic markers induced by conditioned media from IFNγ-stimulated mixed glia and by treatment of neuronal cells with QUIN (1µM). Overall, this study supports a role for the KP in driving neuronal atrophy associated with reactive glia and indicates that inhibition of the KP in glia, or raising the concentration of the astrocytic metabolite KYNA, protects against reactive microglial and QUIN-associated neuronal atrophy.

Astrocytes resolve ER stress through mitochondrial fusion facilitated by biotin availability.

Structures of cellular organelles are intertwined with their functions that undergo alterations once the organelles are stressed. Since organelle functions are dependent on each other, an organelle-specific stress possibly influences the structure and function of its associated organelles. In this perspective, our study demonstrated that endoplasmic reticulum (ER)-specific stress induced by tunicamycin in primary astroglial culture is associated with altered mitochondrial dynamics and matched with the changes as observed in the aging rat brain. However, the exogenous addition of biotin, a highly lipogenic and mitochondrial vitamin, ameliorates ER stress even though its direct targets are not known within ER. Alternatively, the increased biotinylation of mitochondrial carboxylases preserves its basal respiratory capacity by upregulating mitofusin 2 (Mfn2) and, possibly, its associated role on mitochondrial fusion. Furthermore, the Mfn2 increase by biotin augments physical interaction between ER and functional mitochondria to exchange biomolecules as a part of ER stress resolution. This suggests an increased demand for micronutrient biotin under ER stress resolves the same by undergoing appropriate structural and metabolic contacts between ER and mitochondria. These findings provide a paradigm to resolve stress in one organelle by sustaining the metabolic commitments of another interdependent organelle. The findings also highlight a novel role of biotin in inducing Mfn2 expression and localization under ER stress in addition to its known role as a co-enzyme.

Type 1 Interleukin-4 Signaling Obliterates Mouse Astroglia in vivo but Not in vitro

Recent findings suggest that reduced neurogenesis could be one of the underlying reasons for the exacerbated neuropathology in humans, thus restoring the neural stem cell proliferation and neurogenesis could help to circumvent some pathological aspects of Alzheimer’s disease. We recently identified Interleukin-4/STAT6 signaling as a neuron–glia crosstalk mechanism that enables glial proliferation and neurogenesis in adult zebrafish brain and 3D cultures of human astroglia, which manifest neurogenic properties. In this study, by using single cell sequencing in the APP/PS1dE9 mouse model of AD, we found that IL4 receptor (Il4r) is not expressed in mouse astroglia and IL4 signaling is not active in these cells. We tested whether activating IL4/STAT6 signaling would enhance cell proliferation and neurogenesis in healthy and disease conditions. Lentivirus-mediated expression of IL4R or constitutively active STAT6VT impaired the survival capacity of mouse astroglia in vivo but not in vitro. These results suggest that the adult mouse brain generates a non-permissive environment that dictates a negative effect of IL4 signaling on astroglial survival and neurogenic properties in contrast to zebrafish brains and in vitro mammalian cell cultures. Our findings that IL4R signaling in dentate gyrus (DG) of adult mouse brain impinges on the survival of DG cells implicate an evolutionary mechanism that might underlie the loss of neuroregenerative ability of the brain, which might be utilized for basic and clinical aspects for neurodegenerative diseases.

Simvastatin, edaravone and dexamethasone protect against kainate-induced brain endothelial cell damage

BackgroundExcitotoxicity is a central pathological pathway in many neurological diseases with blood–brain barrier (BBB) dysfunction. Kainate, an exogenous excitotoxin, induces epilepsy and BBB damage in animal models, but the direct effect of kainate on brain endothelial cells has not been studied in detail. Our aim was to examine the direct effects of kainate on cultured cells of the BBB and to test three anti-inflammatory and antioxidant drugs used in clinical practice, simvastatin, edaravone and dexamethasone, to protect against kainate-induced changes.MethodsPrimary rat brain endothelial cell, pericyte and astroglia cultures were used to study cell viability by impedance measurement. BBB permeability was measured on a model made from the co-culture of the three cell types. The production of nitrogen monoxide and reactive oxygen species was followed by fluorescent probes. The mRNA expression of kainate receptors and nitric oxide synthases were studied by PCR.ResultsKainate damaged brain endothelial cells and made the immunostaining of junctional proteins claudin-5 and zonula occludens-1 discontinuous at the cell border indicating the opening of the barrier. The permeability of the BBB model for marker molecules fluorescein and albumin and the production of nitric oxide in brain endothelial cells were increased by kainate. Simvastatin, edaravone and dexamethasone protected against the reduced cell viability, increased permeability and the morphological changes in cellular junctions caused by kainate. Dexamethasone attenuated the elevated nitric oxide production and decreased the inducible nitric oxide synthase (NOS2/iNOS) mRNA expression increased by kainate treatment.ConclusionKainate directly damaged cultured brain endothelial cells. Simvastatin, edaravone and dexamethasone protected the BBB model against kainate-induced changes. Our results confirmed the potential clinical usefulness of these drugs to attenuate BBB damage.

A Simple and Efficient Method for Concomitant Isolation and Culture of Enriched Astroglial and Microglial Cells from the Rat Spinal Cord.

Investigations into glial biology have contributed substantially in understanding the physiology and pathology of the nervous system. However, intricacies of the neuron-glial and glial-glial interactions in vivo present significant challenges while delineating the individual cell-type contributions, thus making the in vitro techniques exceedingly relevant to study glial biology. However, obtaining optimal yield along with high purity has been challenging for microglial cultures. Here we present a simple protocol to establish enriched astroglial as well as microglial cultures from the neonatal rat spinal cord. This method results in highly enriched astroglial and microglial cultures with maximal yield.

Metformin mediates neuroprotection and attenuates hearing loss in experimental pneumococcal meningitis

BackgroundPneumococcal meningitis is associated with high risk of neurological sequelae such as cognitive impairment and hearing loss. These sequelae are due to parenchymal brain and inner ear damage primarily induced by the excessive inflammatory reaction in response to bacterial brain invasion. Metformin—a biguanide drug to treat diabetes mellitus type 2—was recently found to suppress neuroinflammation and induce neuroregeneration. This study evaluated the effect of metformin adjunctive to antibiotics on neuroinflammation, brain and inner ear damage, and neurofunctional outcome in experimental pediatric pneumococcal meningitis.MethodsEleven-day-old Wistar rats were infected intracisternally with 5.22 ± 1.27 × 103 CFU Streptococcus pneumoniae and randomized for treatment with metformin (50 mg/kg, i.p., once daily for 3 weeks) plus ceftriaxone (100 mg/kg, i.p., bid, n = 61) or ceftriaxone monotherapy (n = 79). Cortical damage and hippocampal apoptosis were evaluated histomorphometrically 42 h post infection. Cerebrospinal fluid cytokine levels were analyzed during acute infection. Five weeks post infection, auditory brainstem responses were measured to determine hearing thresholds. Spiral ganglion neuron density and abundance of recently proliferated and integrated hippocampal granule neurons were assessed histologically. Additionally, the anti-inflammatory effect of metformin was studied in primary rat astroglial cells in vitro.ResultsUpon pneumococcal infection, metformin treatment significantly reduced levels of inflammatory cytokines and nitric oxide production in cerebrospinal fluid and in astroglial cell cultures in vitro (p < 0.05). Compared to animals receiving ceftriaxone monotherapy, adjunctive metformin significantly reduced cortical necrosis (p < 0.02) during acute infection and improved median click-induced hearing thresholds (60 dB vs. 100 dB, p < 0.002) 5 weeks after infection. Adjuvant metformin significantly improved pure tone hearing thresholds at all assessed frequencies compared to ceftriaxone monotherapy (p < 0.05) and protected from PM-induced spiral ganglion neuron loss in the inner ear (p < 0.05).ConclusionAdjuvant metformin reduces brain injury during pneumococcal meningitis by decreasing the excessive neuroinflammatory response. Furthermore, it protects spiral ganglion neurons in the inner ear and improves hearing impairments after experimental pneumococcal meningitis. These results identify adjuvant metformin as a promising therapeutic option to improve the outcome after pediatric pneumococcal meningitis.

Histone H1 improves regeneration after mouse spinal cord injury and changes shape and gene expression of cultured astrocytes

We have shown that histone H1 is a binding partner for polysialic acid (PSA) and that it improves functional recovery, axon regrowth/sprouting, and target reinnervation after mouse femoral nerve injury. Here, we analyzed whether histone H1 affects functional recovery, axon regrowth/sprouting, and target reinnervation after spinal cord injury of adult mice. Furthermore, we tested in vitro histone H1's effect on astrocytic gene expression, cell shape and migration as well as on cell survival of cultured motoneurons. We applied histone H1 to compressed spinal cord and determined functional recovery and number of fibrillary acidic protein (GFAP)- and neuron-glial antigen 2 (NG2)- positive glial cells, which contribute to glial scarring. Histone H1's effect on migration of astrocytes, astrocytic gene expression and motoneuronal survival was determined using scratch-wounded astroglial monolayer cultures, astrocyte cultures for microarray analysis, and motoneuron cell culture under oxidative stress conditions, respectively. Histone H1 application improves locomotor functions and enhances monoaminergic and cholinergic reinnervation of the spinal cord. Expression levels of GFAP and NG2 around the lesion site were decreased in histone H1-treated mice relative to vehicle-treated mice six weeks after injury. Histone H1 reduced astrocytic migration, changed the shape of GFAP- and NG2-positive glial cells and altered gene expression. Gene ontology enrichment analysis indicated that in particular genes coding for proteins involved in proliferation, differentiation, migration and apoptosis are dysregulated. The up- and down-regulation of distinct genes was confirmed by qPCR and Western blot analysis. Moreover, histone H1 reduced hydrogen peroxide-induced cell death of cultured motoneurons. The combined observations indicate that histone H1 locally applied to the lesion site, improves regeneration after spinal cord injury. Some of these beneficial functions of histone H1 in vivo and in vitro can be attributed to its interaction with PSA-carrying neural cell adhesion molecule.

Mitochondrial biogenesis is altered in HIV+ brains exposed to ART: Implications for therapeutic targeting of astroglia

The neuropathogenesis of HIV associated neurocognitive disorders (HAND) involves disruption of mitochondrial homeostasis and increased neuroinflammation. However, it is unknown if alterations in mitochondrial biogenesis in the brain underlie the neuropathogenesis of HAND. In this study, neuropathological and molecular analyses of mitochondrial biogenesis and inflammatory pathways were performed in brain specimens from a well-characterized cohort of HIV+ cases that were on antiretroviral regimens. In vitro investigations using primary human astroglia and neurons were used to probe the underlying mechanisms of mitochondrial alterations. In frontal cortices from HAND brains compared to cognitive normal brains, total levels of transcription factors that regulate mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were decreased. Immunohistochemical analyses revealed that TFAM was decreased in neurons and increased in astroglia. These changes were accompanied by decreased total mitochondrial DNA per cell and increased levels of messenger RNA for the proinflammatory cytokine interleukin (IL)-1β. To determine how IL-1β affects astroglial bioenergetic processes and mitochondrial activity, human astroglial cultures were exposed to recombinant IL-1β. IL-1β induced mitochondrial activity within 30 min of treatment, altered mitochondrial related gene expression, altered mitochondrial morphology, enhanced adenoside triphosphate (ATP) utilization and increased the expression of inflammatory cytokines. WIN55,212-2 (WIN), an aminoalkylindole derivative and cannabinoid receptor agonist, blocked IL-1β-induced bioenergetic fluctuations and inflammatory gene expression in astroglia independent of cannabinoid receptor (CB)1 and peroxisome proliferator-activated receptor (PPAR) γ. A PPARα antagonist reversed the anti-inflammatory effects of WIN in human astroglia. These results show that mitochondrial biogenesis is differentially regulated in neurons and astroglia in HAND brains and that targeting astroglial bioenergetic processes may be a strategy to modulate neuroinflammation.

Transient Improvement of Cerebellar Oligodendroglial Development in a Neonatal Hyperoxia Model by PDGFA Treatment.

In preterm infants, the changes from fetal life to ex-utero conditions often coincide with reduced growth and white matter damage of the cerebellum. The premature increase in arterial oxygen tension caused by preterm birth may dysregulate cerebellar development. In a hyperoxia rat model of white matter damage to mimic a steep increase in oxygen levels by 24h exposure to 80% O2 from postnatal day 6 (P6) to day 7, we analyzed growth factor (GF) synthesis of cerebellar astrocytes. Determination of GF production was performed in astrocytes after Magnetic-activated cell sorting (MACS) isolation from cerebelli after hyperoxia exposure ex vivo, and also in astroglial cultures. Oligodendrocyte progenitor cell (OPC) function was analyzed in cerebellar OPCs isolated by MACS after hyperoxia. Administration of PDGFA from P6 to P11, during hyperoxia and during 4days recovery, was finally tested for protection of oligodendroglia and myelination. As a result, expression of the GFs Pdgfa, Fgf2, and Bdnf was diminished in cerebellar astrocytes in vitro and in vivo. Gene expression of Olig1, Olig2, Sox9, Sox10, and Cnp was reduced in OPCs in vivo. Nasal PDGFA application improved oligodendroglial proliferation after hyperoxia at P7. However, this treatment effect vanished until P9. Impaired MBP expression after hyperoxia was attenuated by PDGFA treatment until P11, but not beyond when PDGFA supply was stopped. In this study on neonatal cerebellar injury, it is documented for the first time that improvement of oligodendroglial proliferation and of myelination can be achieved by PDGFA treatment. However, the treatment benefit is not maintained long term.