Rat Astrocytes Research Articles

An assessment of the transport mechanism and intraneuronal stability of L-carnosine.

L-Carnosine (β-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H+-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119μM and a maximum velocity of 0.289nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10-4nmol/mg (prot)/min (with 119μM concentration in the medium)-significantly less than active transport speed. However, carnosine concentrations over 12.5mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine.

Feasibility of Photodynamic Therapy for Glioblastoma with the Mitochondria-Targeted Photosensitizer Tetramethylrhodamine Methyl Ester (TMRM).

One of the most challenging problems in the treatment of glioblastoma (GBM) is the highly infiltrative nature of the disease. Infiltrating cells that are non-resectable are left behind after debulking surgeries and become a source of regrowth and recurrence. To prevent tumor recurrence and increase patient survival, it is necessary to cleanse the adjacent tissue from GBM infiltrates. This requires an innovative local approach. One such approach is that of photodynamic therapy (PDT) which uses specific light-sensitizing agents called photosensitizers. Here, we show that tetramethylrhodamine methyl ester (TMRM), which has been used to asses mitochondrial potential, can be used as a photosensitizer to target GBM cells. Primary patient-derived GBM cell lines were used, including those specifically isolated from the infiltrative edge. PDT with TMRM using low-intensity green light induced mitochondrial damage, an irreversible drop in mitochondrial membrane potential and led to GBM cell death. Moreover, delayed photoactivation after TMRM loading selectively killed GBM cells but not cultured rat astrocytes. The efficacy of TMRM-PDT in certain GBM cell lines may be potentiated by adenylate cyclase activator NKH477. Together, these findings identify TMRM as a prototypical mitochondrially targeted photosensitizer with beneficial features which may be suitable for preclinical and clinical translation.

Astrocytes in the ventral pallidum extinguish heroin seeking through GAT-3 upregulation and morphological plasticity at D1-MSN terminals

GABAergic projections from the nucleus accumbens core to the dorsolateral ventral pallidum are necessary for drug-conditioned cues to initiate relapse-like drug seeking. Astrocytes in the ventral pallidum are situated perisynaptically and regulate GABA transmission through expression of GABA uptake transporters, but whether they are involved in regulating drug seeking is unknown. To determine the contribution of ventral pallidal astrocytes to heroin seeking, we labeled astrocytes in male and female rats with a membrane-bound fluorescent tag and used confocal microscopy to quantify astroglial expression of the GABA transporter GAT-3 and astrocyte synaptic proximity after withdrawal from heroin self-administration and during 15 min of cued heroin seeking. We found that GAT-3 was upregulated in rats that had extinguished heroin seeking, but not in animals that were withdrawn from heroin without extinction training or in rats that extinguished sucrose seeking. When GAT-3 upregulation was reversed using a vivo-morpholino oligo, heroin seeking was restored in the extinguished context and extinction of cued heroin seeking was disrupted compared to control animals. Although astrocyte synaptic proximity was not altered overall after heroin withdrawal, examination of astrocyte proximity to accumbens D1- or D2-expressing afferents revealed a selective increase in astrocyte proximity with D1-expressing terminals during extinction of heroin self-administration. Experimentally-induced reduction of astrocyte synaptic proximity through knockdown of the astrocyte-selective actin-binding protein ezrin also markedly disrupted extinction of heroin seeking. Notably, GAT-3 or ezrin knockdown had no impact on context- or cue-induced seeking in sucrose-trained animals. These data show that astrocytes in the ventral pallidum undergo plasticity after extinction of heroin use that reduces seeking and highlight the importance of astrocyte-neuron interactions in shaping behaviors associated with opioid use disorder.

New Blood-Brain Barrier Models Using Primary Parkinson's Disease Rat Brain Endothelial Cells and Astrocytes for the Development of Central Nervous System Drug Delivery Systems.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by motor system defects due to the loss of dopaminergic neurons. A significant contributor to the current limited therapeutic treatments for PD is the poor penetration of potential drugs through the blood-brain barrier (BBB). The BBB is a highly specialized neurovascular system that separates components of the circulating blood from neurons. There is a great need to develop in vitro BBB models that retain fundamental characteristics and reliably predict the permeability of drug candidates. BBB breakdown may initiate and/or contribute to neuronal dysfunction and loss in diseases such as PD. However, there is no in vitro BBB model that mimics the pathological state of PD. To construct in vitro BBB models for drug delivery systems in the developing central nervous system (CNS), we isolated high purity endothelial cells from both normal and PD rat brain microvessels. The primary rat endothelial cell cultures maintained the properties of their in vivo counterparts. We developed and characterized in vitro rat endothelial cell and C6 glial cell coculture BBB models. We further examined the morphological and functional integrity of the barriers. The in vitro coculture BBB models we established displayed the typical cytoarchitecture and cellular markers by immunofluorescence staining and electron microscopy, high transendothelial electrical resistance (>300 Ω cm2), and a low permeability value (<3 × 10-6 cm/s). Our new models can be used to study BBB dysfunctions in relation to the pathogenesis and progression of PD, as well as a screening tool to test candidate drugs for PD treatment.

Novel insights into antidepressant mechanism of Kai Xin San formula: Inhibiting NLRP3 inflammasome activation by promoting autophagy

BackgroundKai Xin San (KXS) was widely applied for the treatment of depression for thousands of years. However, the underlying antidepressant mechanism of KXS remains not clear. PurposeThis study aimed to investigate whether NLRP3 inflammasome and autophagy are involved in inflammation-induced depression and antidepressant mechanism of KXS. MethodsWistar rats were exposed to chronic unpredictable mild stress (CUMS) for 6 weeks, and KXS (3, 5, and 10 g/kg/d) was administrated during the last 2 weeks of CUMS procedure. The effects of KXS on depressive-like behaviors, neuroinflammation, NLRP3 inflammasome activation, and autophagy were investigated in CUMS rats. Rat astrocytes were employed to further explore the potential mechanism of KXS in regulating NLRP3 inflammasome and autophagy. Autophagy inhibitor 3-methyladenine (3-MA, 5 mM) was used in vitro to elucidate the role of autophagy in the antidepressant mechanism of KXS. ResultsIn vivo, KXS improved depressive-like behaviors of CUMS rats in sucrose preference test, open field test and forced swimming test. Moreover, KXS inhibited the neuroinflammation induced by CUMS and promoted autophagy in prefrontal cortex of rats. The results in vitro further validated the anti-inflammatory and proautohapgic effects of KXS. More importantly, autophagy inhibitor 3-MA diminished the inhibitory effect of KXS on NLRP3 inflammasome activation in rat astrocytes. ConclusionKXS ameliorated CUMS-induced depressive behaviors in rats and inhibited the NLRP3 inflammasome-mediated inflammation in vivo and in vitro. These effects might be regulated by KXS-induced autophagy.

Functional Assessment of Stroke-Induced Regulation of miR-20a-3p and Its Role as a Neuroprotectant

MicroRNAs have gained popularity as a potential treatment for many diseases, including stroke. This study identifies and characterizes a specific member of the miR-17–92 cluster, miR-20a-3p, as a possible stroke therapeutic. A comprehensive microRNA screening showed that miR-20a-3p was significantly upregulated in astrocytes of adult female rats, which typically have better stroke outcomes, while it was profoundly downregulated in astrocytes of middle-aged females and adult and middle-aged males, groups that typically have more severe stroke outcomes. Assays using primary human astrocytes and neurons show that miR-20a-3p treatment alters mitochondrial dynamics in both cell types. To assess whether stroke outcomes could be improved by elevating astrocytic miR-20a-3p, we created a tetracycline (Tet)-induced recombinant adeno-associated virus (rAAV) construct where miR-20a-3p was located downstream a glial fibrillary acidic protein promoter. Treatment with doxycycline induced miR-20-3p expression in astrocytes, reducing mortality and modestly improving sensory motor behavior. A second Tet-induced rAAV construct was created in which miR-20a-3p was located downstream of a neuron-specific enolase (NSE) promoter. These experiments demonstrate that neuronal expression of miR-20a-3p is vastly more neuroprotective than astrocytic expression, with animals receiving the miR-20a-3p vector showing reduced infarction and sensory motor improvement. Intravenous injections, which are a therapeutically tractable treatment route, with miR-20a-3p mimic 4 h after middle cerebral artery occlusion (MCAo) significantly improved stroke outcomes including infarct volume and sensory motor performance. Improvement was not observed when miR-20a-3p was given immediately or 24 h after MCAo, identifying a unique delayed therapeutic window. Overall, this study identifies a novel neuroprotective microRNA and characterizes several key pathways by which it can improve stroke outcomes.

Use of Cocultures to Measure the Blood-Brain Barrier Permeability of Oxytocin.

Primary monkey brain capillary endothelial cell cultures, with rat pericytes and astrocytes, provide an assay system for predicting the ability of oxytocin (OT) to cross the blood-brain barrier (BBB), using a commercially available in vitro BBB kit. The integrity of the in vitro "BBB," which has a high transendothelial electrical resistance (TEER), can be established approximately 4days after preparations for experiments. Dominant endothelial transport of OT is from the upper (luminal blood side) to lower (abluminal brain side) chambers, dose-dependently. OT is transported by the receptor for advanced glycation end-products (RAGE) in endothelial cells, which is evidenced using the RAGE knockdown system with short hairpin RNA (shRNA) treatment. This in vitro assay system is useful for further assessment of OT transport across the BBB.

Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes.

Culture models of the blood-brain barrier (BBB) are important research tools. Their role in the preclinical phase of drug development to estimate the permeability for potential neuropharmaceuticals is especially relevant. Since species differences in BBB transport systems exist, primate models are considered as predictive for drug transport to brain in humans. Based on our previous expertise we have developed and characterized a non-human primate co-culture BBB model using primary cultures of monkey brain endothelial cells, rat brain pericytes, and rat astrocytes. Monkey brain endothelial cells in the presence of both pericytes and astrocytes (EPA model) expressed enhanced barrier properties and increased levels of tight junction proteins occludin, claudin-5, and ZO-1. Co-culture conditions also elevated the expression of key BBB influx and efflux transporters, including glucose transporter-1, MFSD2A, ABCB1, and ABCG2. The correlation between the endothelial permeability coefficients of 10 well known drugs was higher (R2 = 0.8788) when the monkey and rat BBB culture models were compared than when the monkey culture model was compared to mouse in vivo data (R2 = 0.6619), hinting at transporter differences. The applicability of the new non-human primate model in drug discovery has been proven in several studies.

Reactive astrocytes induced by 2-chloroethanol modulate microglia polarization through IL-1β, TNF-α, and iNOS upregulation

The synthetic organic chemical, 1,2-dichloroethane (1,2-DCE), can cause brain edemas under subacute poisoning. Our previous studies indicated that neuroinflammation could be induced due to astrocytes and microglia activation during brain edemas in 1,2-DCE-intoxicated mice. However, the crosstalk between these two glial cells in 1,2-DCE-induced neuroinflammation remained unclear. In this study, primary cultured rat astrocytes and microglia, as well as an immortalized microglia cell line were employed to study the effects of 2-chloroethanol (2-CE, a 1,2-DCE intermediate metabolite in vivo) treated astrocytes on microglia polarization. Our current results revealed that 2-CE treated rat astrocytes were activated through p38 mitogen-activated protein kinase (p38 MAPK)/nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) signaling pathways. Theses pathways were triggered by reactive oxygen species (ROS) produced during 2-CE metabolism. Also, astrocytes were more sensitive to 2-CE effects than microglia. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) expressions were upregulated in 2-CE-induced reactive astrocytes, enhancing IL-1β, TNF-α, and nitric oxide (NO) excretions, which stimulated microglia polarization. Therefore, the neuroinflammation induced by 1,2-DCE in mice's brains is probably triggered by reactive astrocytes.

Reprogramming rat astrocytes into neurons using small molecules for cell replacement following intracerebral hemorrhage

Astrocytes are promising source cells to replace neurons lost to disease owing to a shared lineage and capacities for dedifferentiation and proliferation under pathological conditions. Reprogramming of astrocytes to neurons has been achieved by transcription factor modulation, but reprogramming in vitro or in vivo using small‐molecule drugs may have several advantages for clinical application. For instance, small molecules can be extensively characterized for efficacy, toxicity, and tumorigenicity in vitro; induce rapid initiation and subsequent reversal of transdifferentiation upon withdrawal, and obviate the need for exogenous gene transfection. Here we report a new astrocyte–neuron reprogramming strategy using a combination of small molecules (0.5 mM valproic acid, 1 μM RepSox, 3 μM CHIR99021, 2 μM I‐BET151, 10 μM ISX‐9, and 10 μM forskolin). Treatment with this drug combination gradually reduced expression levels of astroglial marker proteins (glial fibrillary acidic protein and S100), transiently enhanced expression of the neuronal progenitor marker doublecortin, and subsequently elevated expression of the mature neuronal marker NeuN in primary astrocyte cultures. These changes were accompanied by transition to a neuron‐like morphological phenotype and expression of multiple neuronal transcription factors. Further, this drug combination induced astrocyte‐to‐neuron transdifferentiation in a culture model of intracerebral hemorrhage (ICH) and upregulated many transdifferentiation‐associated signaling molecules in ICH model rats. In culture, the drug combination also reduced ICH model‐associated oxidative stress, apoptosis, and pro‐inflammatory cytokine production. Neurons derived from small‐molecule reprogramming of astrocytes in adult Sprague–Dawley rats demonstrated long‐term survival and maintenance of neuronal phenotype. This small‐molecule‐induced astrocyte‐to‐neuron transdifferentiation method may be a promising strategy for neuronal replacement therapy.

Inhibition of Autophagy Flux Promotes Secretion of Chondroitin Sulfate Proteoglycans in Primary Rat Astrocytes.

Following spinal cord injury (SCI), reactive astrocytes in the glial scar produce high levels of chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal regeneration. Transforming growth factor beta (TGFβ) is a well-known factor that induces the production of CSPGs, and in this study, we report a novel mechanism underlying TGFβ's effects on CSPG secretion in primary rat astrocytes. We observed increased TGFβ-induced secretion of the CSPGs neurocan and brevican, and this occurred simultaneously with inhibition of autophagy flux. In addition, we show that neurocan and brevican levels are further increased when TGFβ is administered in the presence of an autophagy inhibitor, Bafilomycin-A1, while they are reduced when cells are treated with a concentration of rapamycin that is not sufficient to induce autophagy. These findings suggest that TGFβ mediates its effects on CSPG secretion through autophagy pathways. They also represent a potential new approach to reduce CSPG secretion in vivo by targeting autophagy pathways, which could improve axonal regeneration after SCI.

Anti-AQP4 autoantibodies promote ATP release from astrocytes and induce mechanical pain in rats

BackgroundIntractable neuropathic pain is a common symptom of neuromyelitis optica spectrum disorder (NMOSD). However, the underlying mechanism of NMOSD pain remains to be elucidated. In this study, we focused on ATP, which is one of the damage-associated molecular patterns, and also a well-recognized molecule involved in peripheral neuropathic pain.MethodsWe assessed the development of pain symptoms by injecting anti-AQP4 recombinant autoantibodies (rAQP4 IgG) into rat spinal cords. We incubated HEK293 cells expressing AQP4 (HEK-AQP4) and rat astrocytes with rAQP4 IgG and assessed the level of ATP in the supernatant. We performed transcriptome analysis of the spinal cords injected with rAQP4 IgG. Pharmacological inhibition was also applied to investigate the involvement of ATP in the development of neuropathic pain in our rat model. The ATP concentration within the cerebrospinal fluid was examined in patients with NMOSD and other neurological diseases.ResultsDevelopment of mechanical allodynia was confirmed in rAQP4 IgG–treated rats. AQP4-Ab–mediated extracellular ATP release from astrocytes was observed in vitro, and pharmacological inhibition of ATP receptor reversed mechanical allodynia in the rAQP4 IgG–treated rats. Furthermore, transcriptome analysis revealed elevation of gene expressions related to several ATP receptors including P2rx4 and IL1B in the spinal cord of rAQP4 IgG–treated rats. In patients, CSF ATP concentration was significantly higher in the acute and remission phase of NMOSD than in multiple sclerosis or other neurological disorders.ConclusionAnti-AQP4 antibody was shown to induce the release of extracellular ATP from astrocytes. The ATP-mediated development of mechanical allodynia was also suggested in rats treated with anti-AQP4 antibody. Our study indicates the pivotal role of ATP in the pain mechanism of NMOSD.

Integrated high-throughput small RNA and transcriptome sequencing unveil the shape-dependent toxicity of nano-alumina in rat astrocytes

BackgroundThe large-scale applications of alumina nanoparticles (Al2O3-NPs), one of the most important NPs in the global market, are causing severe damages to the environment and human health. Our previous research has revealed a critical role of nanoparticle morphology (e.g., flake and rod) in determining the toxic potencies of Al2O3-NPs, where nanorods demonstrated a significantly stronger toxic response than that of nanoflakes. However, their underlying mechanisms have not been completely elucidated yet. In the present study, we evaluated and compared the potential toxicological mechanisms of two shapes of γ-Al2O3-NPs (flake versus rod) by measuring miRNA and mRNA profiles of astrocytes in rat cerebral cortex, ex vivo.ResultsA total of 269 mRNAs and 122 miRNAs, 180 mRNAs and 116 miRNAs were differentially expressed after nanoflakes or nanorods exposure, respectively. Among them, 55 miRNAs (e.g., miR-760-5p, miR-326-3p, and miR-35) and 105 mRNAs (e.g., Kdm4d, Wdr62, and Rps6) showed the same trend between the two shapes. These miRNAs and mRNAs were mainly involved in apoptosis, inflammatory pathways (e.g., NF-kappa B), carcinogenic pathways (e.g., MAPK, p53, Notch, Rap1, and Ras), and cellular lipid metabolisms (e.g., glycerolipid metabolism, sphingolipid, and ether lipid metabolism). However, the remaining miRNAs and mRNAs either showed an opposite trend or only changed by a particular shape. Nanorods could specifically alter the changes of PI3K/Akt, AMPK and TNF pathways, cell cycle, and cellular senescence, while nanoflakes caused the changes of Toll and lmd signaling pathways.ConclusionsCombined with previous research results, we further revealed the potential biomolecular mechanisms leading to the stronger toxicity of nanorods than that of nanoflakes, and multi-omics is a powerful approach to elucidate morphology-related mode of actions.

Overexpression of miR-221-3p effecting cell proliferation, apoptosis and inflammation by targeting Toll-like receptors 4 in propofol-induced rat astrocytes

IntroductionGrowing evidence indicated that propofol has neurotoxic effects on the brains of developing rodents, leading to neuronal cell death, neurodegeneration, and brain injury.Material and methodsEctopic miR-221-3p was transfected into rat astrocytes, and Cell Counting Kit-8 assay and flow cytometry were performed to evaluate cell growth and apoptosis. The mRNA levels of Toll-like receptors 4 (TLR4), nuclear factor kappa B, interleukin-6, interleukin-1β, myeloid differentiation primary response 88 (MyD88), caspase-3, caspase-12, STAT3, and GRP78 were detected using quantitative real-time polymerase chain reaction. The proteins of TLR4 and MyD88 were determined using Western blotting. The association between miR-221-3p and TLR4 was measured using Dual-Luciferase Reporter Assay (Promega Corporation, Wisconsin, USA). Then, siTLR4 was transfected with 293T cells to study the role of TLR4 in astrocytes with propofol treatment.ResultsThe miR-221-3p expression in rat astrocytes was markedly suppressed by propofol treatment. The miR-221-3p mimics transfection in propofol-treated astrocytes effectively reduced the suppressive effect of propofol on astrocyte growth, repressed the propofol-induced apoptosis in rat astrocytes, and decreased the cell number during the G2–M phase. The expression of MyD88 and TLR4 was induced by propofol, whereas the transfection of miR-221-3p mimics dramatically reduced these genes expression at the mRNA and protein expression. After that, TLR4 was found to be target of miR-221-3p using Dual-Luciferase Reporter Assay. Furthermore, knockdown of TLR4 could suppress the apoptosis rate in propofol-treated astrocytes.ConclusionsThis study revealed that miR-221-3p might prevent astrocytes from propofol-induced damage by targeting TLR4.

Neuronal chemokine-like-factor 1 (CKLF1) up-regulation promotes M1 polarization of microglia in rat brain after stroke.

The phenotypic transformation of microglia in the ischemic penumbra determines the outcomes of ischemic stroke. Our previous study has shown that chemokine-like-factor 1 (CKLF1) promotes M1-type polarization of microglia. In this study, we investigated the cellular source and transcriptional regulation of CKLF1, as well as the biological function of CKLF1 in ischemic penumbra of rat brain. We showed that CKLF1 was significantly up-regulated in cultured rat cortical neurons subjected to oxygen-glucose deprivation/reoxygenation (ODG/R) injury, but not in cultured rat microglia, astrocytes and oligodendrocytes. In a rat model of middle cerebral artery occlusion, we found that CKLF1 was up-regulated and co-localized with neurons in ischemic penumbra. Furthermore, the up-regulated CKLF1 was accompanied by the enhanced nuclear accumulation of NF-κB. The transcriptional activity of CKLF1 was improved by overexpression of NF-κB in HEK293T cells, whereas application of NF-κB inhibitor Bay 11-7082 (1 μM) abolished it, caused by OGD/R. By using chromatin-immunoprecipitation (ChIP) assay we demonstrated that NF-κB directly bound to the promoter of CKLF1 (at a binding site located at -249 bp to -239 bp of CKLF1 promoter region), and regulated the transcription of human CKLF1. Moreover, neuronal conditional medium collected after OGD/R injury or CKLF1-C27 (a peptide obtained from secreted CKLF1) induced the M1-type polarization of microglia, whereas the CKLF1-neutralizing antibody (αCKLF1) or NF-κB inhibitor Bay 11-7082 abolished the M1-type polarization of microglia. Specific knockout of neuronal CKLF1 in ischemic penumbra attenuated neuronal impairments and M1-type polarization of microglia caused by ischemic/reperfusion injury, evidenced by inhibited levels of M1 marker CD16/32 and increased expression of M2 marker CD206. Application of CKLF1-C27 (200 nM) promoted the phosphorylation of p38 and JNK in microglia, whereas specific depletion of neuronal CKLF1 in ischemic penumbra abolished ischemic/reperfusion-induced p38 and JNK phosphorylation. In summary, CKLF1 up-regulation in neurons regulated by NF-κB is one of the crucial mechanisms to promote M1-type polarization of microglia in ischemic penumbra.

Onjisaponin B attenuates glutamate release via inhibition of calmodulin-dependent protein kinase II and connexin 43 pathways in rat astrocytes subjected to oxygen and glucose deprivation

Background: Post-stroke depression (PSD) is one of the most common complications of stroke. Many studies have confirmed that PSD is associated with the abnormal release of glutamic acid (Glu) via the Ca2+/calmodulin (CaM)/CaM-dependent protein kinase II (CaMKII) and connexin 43 (Cx43) hemichannel pathways. Onjisaponin B, a traditional Chinese medicine, has been discovered to have neuroprotective effects. Objectives: In this study, we aimed to investigate the effects and underlying mechanisms of onjisaponin B on the release of glutamate in rat astrocytes subjected to oxygen and glucose deprivation (OGD). Materials and Methods: The ischemic and anoxic cell model was established in rat astrocytes through OGD injury. Rat astrocytes were randomly divided into control, model, 10 μM onjisaponin B, and 20 μM onjisaponin B groups. Cell viability was assessed by acridine orange/ethidium bromide bilabel assay. The intracellular Ca2+ level was measured with Fluo-3-AM as the fluorescence indicator. Western blot analysis and quantitative polymerase chain reaction were used to detect the expressions of Cx43 and CaMKII in the samples. The glutamate level of the extracellular fluid was determined using high-performance liquid chromatography-mass spectrometry. Results: The intracellular Ca2+ levels and the mRNA and protein expression of CaMKII and Cx43 in the onjisaponin B group were significantly lower than that of the model group. The glutamate level in the extracellular fluid was significantly reduced by onjisaponin B in comparison to that in the model group. Moreover, onjisaponin B attenuated the inhibitory effect of OGD on the cell viability of astrocytes. Conclusion: The results of this study suggest that onjisaponin B reduced the release of glutamate via inhibition of the Ca2+/CaMKII and Cx43 pathways in the rat astrocytes, thus inhibiting the downstream glutamic pathway and reinforcing the cell viability of astrocyte.

Retraction.

Retraction: "Effects of microRNA-206 and its target gene IGF-1 on sevoflurane-induced activation of hippocampal astrocytes in aged rats through the PI3K/AKT/CREB signaling pathway," by Tie-Jun Liu, Bin Wang, Qun-Xi Li, Xiao- Liu Dong, Xiao-Liang Han, Shu-Bo Zhang, J Cell Physiol. 2018; 4294-4306: The above article, published online on 20 October 2017 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcp.26248) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Gregg Fields, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. Several flaws and inconsistencies between results presented and experimental methods described were found. The editors consider the conclusions of this article to be invalid.

Differential production of interleukin‐6 and tumor necrosis factor‐α in primary rat astrocyte cultures using two distinct methods of microglia elimination

AbstractHepatic encephalopathy (HE) is a neurocognitive disorder due to acute or chronic liver failure as well as portosystemic bypass. Both neuroinflammation and oxidative stress have been strongly implicated in the pathogenesis of HE. An increasing body of evidence suggests that in the development of these mechanisms astrocytes may be involved. With regard of HE, this aspect of astrocytes is, however, neglected. While several studies focus on astrocytes related to the above‐mentioned pathological processes, the applied in vitro models are generated by diverse methods, which make the results difficult to compare with each other. In addition, these astrocyte cultures frequently contain microglia as well, complicating the interpretation of results. Therefore, we applied enriched primary rat astrocyte cultures generated by microglia elimination via shaking or the combination of cytosine β‐d‐arabinofuranoside hydrochloride (AraC) and l‐leucine methyl ester (LME). We examine whether some HE‐relevant pathological factors, such as ammonia, manganese (Mn), lipopolysaccharide (LPS), and H2O2 could elicit oxidative stress and cytokine production in astrocytes. Mn exposure increased the oxidative stress in astrocytes in a time‐dependent manner; furthermore, we could detect tumor necrosis factor (TNF)‐α production in both cultures at resting state. In addition, it was around 10‐fold higher in the cultures after shaking. LPS increased TNF‐α secretion in cultures after shaking, but not in cultures after AraC + LME treatment. In contrast, interleukin (IL)‐6 was detectable only in the AraC + LME‐treated cultures. In conclusion, we have demonstrated that HE‐relevant factors evoke oxidative stress and cytokine production, albeit depending on the elimination method used.

Baicalein Delays H2O2-Induced Astrocytic Senescence through Inhibition of Senescence-Associated Secretory Phenotype (SASP), Suppression of JAK2/STAT1/NF-κB Pathway, and Regulation of Leucine Metabolism.

Baicalein is an active ingredient extracted from the dried roots of the Scutellaria baicalensis Georgi. It has been demonstrated to improve memory impairment in multiple animal models; however, the underlying mechanisms remain ambiguous. The accumulation of senescent astrocytes and senescence-associated secretory phenotype (SASP) secreted by senescent astrocytes has been deemed as potential contributors to neurodegenerative diseases. Therefore, this study explored the protective effects of baicalein against astrocyte senescence and investigated the molecular mechanisms and metabolic mechanisms of baicalein against astrocyte senescence. Our results demonstrated that treatment with baicalein protects T98G cells from H2O2-induced damage, delays cell senescence, inhibits the secretion of SASP (IL-6, IL-8, TNF-α, CXCL1, and MMP-1), and inhibits SASP-related pathways NF-κB and JAK2/STAT1. 1H NMR metabolomics analysis and correlation analysis revealed that leucine was significantly correlated with SASP factors. Further study demonstrated that supplement with leucine could restrain SASP secretion, and baicalein could significantly increase leucine level through down-regulation of BCAT1 and up-regulation of SLC7A5 expression. The above results revealed that baicalein exerted protective and antisenescence effects in H2O2-induced T98G cells possibly through inhibition of SASP, suppression of JAK2/STAT1/NF-κB pathway, and regulation of leucine metabolism. Consistent results were obtained in primary astrocytes of newborn SD rats, which suggests that baicalein significantly increases viabilities, delays senescence, inhibits IL-6 secretion, and increases leucine level in H2O2-induced primary astrocytes.

Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology.

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging‐related Alzheimer's disease (AD)‐like pathology. However, concerns over adverse effects have slowed the transition of common CN‐inhibiting drugs to the clinic for the treatment of AD and AD‐related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN‐mediated dephosphorylation of a non‐NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild‐type mice (3–4 months old) infused with oligomeric Aβ peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild‐type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging‐related disorders.