Primary Rat Cortical Cells Research Articles

Comparison of the neurotoxic potency of different ultrafine particle fractions from diesel engine exhaust following direct and simulated inhalation exposure

Exposure to traffic-related air pollution and ultrafine particles (<100 nm; UFP) is linked with neurodegeneration. However, the impact of the aromatic content in fuels and the contribution of different fractions of UFP, i.e., solid UFP vs SVOC UFP, on neuronal function is unknown.We therefore studied effects on neuronal activity and viability in rat primary cortical cells exposed for up to 120 h to copper oxide particles (CuO) or UFP (solid and SVOC) emitted from a heavy-duty diesel engine fueled with petroleum diesel (A20; 20 % aromatics) or Hydrotreated Vegetable Oil-type fuel (A0; 0.1 % aromatics), or solid UFP emitted from a non-road Kubota engine fueled with A20.Moreover, effects of UFP and CuO upon simulated inhalation exposure were studied by exposing an lung model (Calu-3 and THP-1 cells) for 48 h and subsequently exposing the cortical cells to the medium collected from the basal compartment of the lung model. Additionally, cell viability, cytotoxicity, barrier function, inflammation, and oxidative and cell stress were studied in the lung model after 48 h exposure to UFP and CuO.Compared to control, direct exposure to CuO and SVOC UFP decreased neuronal activity, which was partly associated with cytotoxicity. Effects on neuronal activity upon direct exposure to solid UFP were limited. A20-derived UFP (solid and SVOC) were more potent in altering neuronal function and viability than A0 counterparts. Effects on neuronal activity from simulated inhalation exposure were minor compared to direct exposures. In the lung model, CuO and A20-derived UFP increased cytokine release compared to control, whereas CuO and SVOC A20 altered gene expression indicative for oxidative stress.Our data indicate that SVOC UFP exhibit higher (neuro)toxic potency for altering neuronal activity in rat primary cortical cells than the solid fraction. Moreover, our data suggest that reducing the aromatic content in fuel decreases the (neuro)toxic potency of emitted UFP.

Galectin-1 as a marker for microglia activation in the aging brain

Microglia cells, the immune cells residing in the brain, express immune regulatory molecules that have a central role in the manifestation of age-related brain characteristics. Our hypothesis suggests that galectin-1, an anti-inflammatory member of the beta-galactoside-binding lectin family, regulates microglia and neuroinflammation in the aging brain. Through our in-silico analysis, we discovered a subcluster of microglia in the aged mouse brain that exhibited increased expression of galectin-1 mRNA. In our Western blotting experiments, we observed a decrease in galectin-1 protein content in our rat primary cortical cultures over time. Additionally, we found that the presence of lipopolysaccharide, an immune activator, significantly increased the expression of galectin-1 protein in microglial cells. Utilizing flow cytometry, we determined that a portion of the galectin-1 protein was localized on the surface of the microglial cells. As cultivation time increased, we observed a decrease in the expression of activation-coupled molecules in microglial cells, indicating cellular exhaustion. In our mixed rat primary cortical cell cultures, we noted a transition of amoeboid microglial cells labeled with OX42(CD11b/c) to a ramified, branched phenotype during extended cultivation, accompanied by a complete disappearance of galectin-1 expression. By analyzing the transcriptome of a distinct microglial subpopulation in an animal model of aging, we established a correlation between chronological aging and galectin-1 expression. Furthermore, our in vitro study demonstrated that galectin-1 expression is associated with the functional activation state of microglial cells exhibiting specific amoeboid morphological characteristics. Based on our findings, we identify galectin-1 as a marker for microglia activation in the context of aging.

Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro.

Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2'-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional (3D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.

A human iPSC-based in vitro neural network formation assay to investigate neurodevelopmental toxicity of pesticides.

Proper brain development is based on the orchestration of key neurodevelopmental processes (KNDP), including the for­mation and function of neural networks. If at least one KNDP is affected by a chemical, an adverse outcome is expected. To enable a higher testing throughput than the guideline animal experiments, a developmental neurotoxicity (DNT) in vitro testing battery (DNT IVB) comprising a variety of assays that model several KNDPs was set up. Gap analysis revealed the need for a human-based assay to assess neural network formation and function (NNF). Therefore, we established the human NNF (hNNF) assay. A co-culture comprised of human induced pluripotent stem cell (hiPSC)-derived excitatory and inhibitory neurons as well as primary human astroglia was differentiated for 35 days on microelectrode arrays (MEA), and spontaneous electrical activity, together with cytotoxicity, was assessed on a weekly basis after washout of the compounds 24 h prior to measurements. In addition to the characterization of the test system, the assay was challenged with 28 com­pounds, mainly pesticides, identifying their DNT potential by evaluating specific spike-, burst-, and network parameters. This approach confirmed the suitability of the assay for screening environmental chemicals. Comparison of benchmark con­centrations (BMC) with an NNF in vitro assay (rNNF) based on primary rat cortical cells revealed differences in sensitivity. Together with the successful implementation of hNNF data into a postulated stressor-specific adverse outcome pathway (AOP) network associated with a plausible molecular initiating event for deltamethrin, this study suggests the hNNF assay as a useful complement to the DNT IVB.

The interrelationships between neuronal viability, synaptic integrity, microglial responses, and amyloid-beta formation in an in vitro neurotrauma model

The interrelationships between neuronal viability, synaptic integrity, and microglial responses remain in infancy. In dealing with the question, we induced a stretch injury to evaluate the mechanical effects of trauma on rat primary cortical neurons and BV2 microglial cells in a transwell culture system. The viability of primary neurons and BV2 cells was determined by MTT. Synaptic integrity was evaluated by determining the expression of beta-secretase 1 (BACE1), amyloid-beta (Aβ), microtubule-associated protein 2 (MAP2), and synaptophysin (vehicle protein). Both CD16/32-positive (CD16/32+) and CD206-positive (CD206+) microglia cells were detected by immunofluorescence staining. The phagocytic ability of the BV2 cells was determined using pHrodo E. coli BioParticles conjugates and flow cytometry. We found that stretch injury BV2 cells caused reduced viability and synaptic abnormalities characterized by Aβ accumulation and reductions of BACE1, MAP2, and synaptophysin in primary neurons. Intact BV2 cells exhibited normal phagocytic ability and were predominantly CD206+ microglia cells, whereas the injured BV2 cells exhibited reduced phagocytic ability and were predominantly CD16/32+ microglial cells. Like a stretch injury, the injured BV2 cells can cause both reduced viability and synaptic abnormalities in primary neurons; intact BV2 cells, when cocultured with primary neurons, can protect against the stretch-injured-induced reduced viability and synaptic abnormalities in primary neurons. We conclude that CD206+ and CD16/32+ BV-2 cells can produce neuroprotective and cytotoxic effects on primary cortical neurons.

The Cholinergic Pathway and MitoK ATP Induce UCP4 Expression Involved in Neuroprotection of FN Stimulation in Rats

Background Electrical stimulation of the cerebellar fastigial nucleus (FN) reduces the infarct size induced by middle cerebral artery occlusion in rats. FN stimulation confers long‐lasting protection from brain injury; however, its underlying mechanism is not yet understood. We aimed to elucidate the mechanism by which FN stimulation exerts neuroprotection. We hypothesized that the neuroprotective effect of FN stimulation involves activation of cholinergic pathways, which increases reactive oxygen species (ROS) production by opening mitochondrial K + ATP channels, thus leading to an increase in UCP4 (uncoupling protein 4) expression and subsequent neuroprotection. Methods FN stimulation was performed for 1 hour in rats. The UCP4 protein and mRNA levels were measured by western blot, dot blot, and in situ hybridization. Carbachol was applied following UCP4‐promoter tdTomato reporter vector transfection of the rat primary cortical cell culture (in vitro) and rat brain (in vivo). We observed cellular UCP4 expression using fluorescence microscopy. UCP4 expression in the cell culture in response to diazoxide application was determined by a reverse transcription‐polymerase chain reaction and western blotting. Results Whereas FN stimulation increased UCP4 protein and mRNA levels, carbachol administration induced UCP4 expression in vitro and in vivo. The attenuation of this effect by atropine suggests that FN‐induced UCP4 expression involves the cholinergic pathway. The opening of mitochondrial K + ATP channels with diazoxide increased the production of ROS and led to increased UCP4 expression. In contrast, quenching ROS with superoxide dismutase reversed the effect of diazoxide on UCP4 expression. Therefore, the opening of mitochondrial K + ATP channels increased ROS production, which subsequently enhanced UCP4 expression and attenuated ROS generation. Conclusion Neuroprotective effect of FN stimulation involves activation of the cholinergic pathways, which increases ROS production by opening mitochondrial K + ATP channels, leading to increased expression of neuroprotective UCP4.

Acute, sub-chronic and chronic exposures to TiO2 and Ag nanoparticles differentially affects neuronal function in vitro

In vivo toxicokinetic studies provide evidence for the translocation and accumulation of nanoparticles (NP) in the brain, thereby causing concern for adverse health effects, particularly for effects following chronic exposure. To date, only few studies investigated the effects of NP exposure on neuronal function in vitro, primarily focusing on short-term effects. The aim of this study was therefore to investigate the effects of two common types of NP, titanium dioxide NP (TiO2NP) and silver NP (AgNP), on neuronal function following acute (0.5 h), sub-chronic (24 h and 48 h) and chronic (14 days) exposure in vitro. Effects of NP exposure on intracellular calcium homeostasis, spontaneous neuronal (network) activity and neuronal network morphology were investigated in rat primary cortical cells using respectively, single-cell microscopy calcium imaging, micro-electrode array (MEA) recordings and immunohistochemistry. Our data demonstrate that high doses of AgNP (≥ 30 µg/mL) decrease calcium influx after 24 h exposure, although neuronal activity is not affected following acute and sub-chronic exposure. However, chronic exposure to non-cytotoxic doses of AgNP (1–10 µg/mL) potently decreases spontaneous neuronal (network) activity, without affecting network morphology and viability. Exposure to higher doses (≥ 30 µg/mL) affects network morphology and is also associated with cytotoxicity. In contrast, acute and sub-chronic exposure to TiO2NP is without effects, whereas chronic exposure only modestly reduces neuronal function without affecting morphology. Our combined findings indicate that TiO2NP exposure is of limited hazard for neuronal function whereas AgNP, in particularly during chronic exposure, has profound effects on neuronal (network) function and morphology.

Extrusion 3D bioprinting of functional self-supporting neural constructs using a photoclickable gelatin bioink

Many in vitro models of neural physiology utilize neuronal networks established on two-dimensional substrates. Despite the simplicity of these 2D neuronal networks, substrate stiffness may influence cell morphology, network interactions and how neurons communicate and function. With this perspective, three-dimensional (3D) gel encapsulation is a powerful to recapitulating aspects of in vivo features, yet such an approach is often limited in terms of the level of resolution and feature size relevant for modelling aspects of brain architecture. Here, we report 3D bioplotting of rat primary cortical neural cells using a hydrogel system comprising gelatin norbornene (GelNB) and poly (ethylene glycol) dithiol (PEGdiSH). This bioink benefits from a rapid photo-click chemistry, yielding eight-layer crosshatch neural scaffolds and a filament width of 350 µm. The printability of this system depends on hydrogel concentration, printing temperature, extrusion pressure and speed. These parameters were studied via quantitative comparison between rheology and filament dimensions to determine the optimal printing conditions. Under optimal conditions, cell viability of bioprinted primary cortical neurons at day 1 (68 ± 2%) and at day 7 (68 ± 1%) were comparable to the 2D control group (72 ± 7%). The present study relates material rheology and filament dimensions to generate compliant free-standing neural constructs through bioplotting of low-concentration GelNB-PEGdiSH, which may provide a step forward to study 3D neuronal function and network formation.

Dendrobium nobile Lindl. Alkaloids Ameliorate Aβ25-35-Induced Synaptic Deficits by Targeting Wnt/β-Catenin Pathway in Alzheimer's Disease Models.

Dendrobium nobile Lindl. alkaloids (DNLA) are effective in ameliorating cognitive deficit in SAMP8, AβPP/PS1, and LPS-induced AD animal models, and prevented Aβ-induced synaptic degeneration in cultured hippocampal neurons. However, the underlying mechanisms remain unexplored. This study investigated the protective effects of DNLA on synaptic damage in an Aβ25-35-induced rat AD model, in primary cortical neuron cultures, and in PC12 cells transfected with human AβPP695, focusing on the Wnt/β-catenin pathway. Sprague-Dawley rats received a single Aβ25-35 injection (10μg) into the bilateral hippocampi. DNLA (40 and 80 mg/kg/d) was intragastrically administrated 7 days prior to Aβ injection and continued for 28 days. The spatial learning and memory, synaptic morphology, synapse-related proteins, and Wnt signaling components GSK3β and β-catenin phosphorylation were evaluated. Rat primary cortical neuron cultures and AβPP695-PC12 cells were used to evaluate axonal mitochondria distribution, reactive oxygen species production, amyloidogenesis, and Wnt pathway in the protection. DNLA ameliorated Aβ-induced cognitive impairment, increased the number of synapses, elevated the postsynaptic density thickness and expression of synapsin and PSD95 in the hippocampus, and suppressed Aβ-mediated GSK3β activity and the β-catenin phosphorylation. In primary neurons and AβPP695-PC12 cells, DNLA restored Aβ25-35 induced mitochondrial dysfunction and inhibited reactive oxygen species production and amyloidogenesis. Furthermore, the Wnt/β-catenin pathway inhibitor Dkk-1 blocked the effect of DNLA on the expression of Aβ1-42 and PSD95. DNLA rescued Aβ-mediated synaptic and mitochondrial injury and inhibited amyloidogenesis in vivo and in vitro, probably through the activation of Wnt/β-catenin signaling pathway to protect synaptic integrity.

Human pluripotent stem cell-derived ectomesenchymal stromal cells promote more robust functional recovery than umbilical cord-derived mesenchymal stromal cells after hypoxic-ischaemic brain damage.

Aims: Hypoxic-ischaemic encephalopathy (HIE) is one of the most serious complications in neonates and infants. Mesenchymal stromal cell (MSC)-based therapy is emerging as a promising treatment avenue for HIE. However, despite its enormous potential, the clinical application of MSCs is limited by cell heterogeneity, low isolation efficiency and unpredictable effectiveness. In this study, we examined the therapeutic effects and underlying mechanisms of human pluripotent stem cell-derived ectomesenchymal stromal cells (hPSC-EMSCs) in a rat model of HIE.Methods: hPSC-EMSCs were induced from either human embryonic stem cells or induced pluripotent stem cells. Stem cells or the conditioned medium (CM) derived from stem cells were delivered intracranially or intranasally to neonatal rats with HIE. Human umbilical cord-derived MSCs (hUC-MSCs) were used as the therapeutic comparison control and phosphate-buffered saline (PBS) was used as a negative control. Lesion size, apoptosis, neurogenesis, astrogliosis and microgliosis were evaluated. The rotarod test and Morris water maze were used to determine brain functional recovery. The PC-12 cell line, rat primary cortical neurons and neural progenitor cells were used to evaluate neurite outgrowth and the neuroprotective and neurogenesis effects of hPSC-EMSCs/hUC-MSCs. RNA-seq and enzyme-linked immunosorbent assays were used to determine the secretory factors that were differentially expressed between hPSC-EMSCs and hUC-MSCs. The activation and suppression of extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) were characterised using western blotting and immunofluorescent staining.Results: hPSC-EMSCs showed a higher neuroprotective potential than hUC-MSCs, as demonstrated by a more significant reduction in lesion size and apoptosis in the rat brain following hypoxia-ischaemia (HI). Compared with PBS treatment, hPSC-EMSCs promoted endogenous neurogenesis and alleviated astrogliosis and microgliosis. hPSC-EMSCs were more effective than hUC-MSCs. hPSC-EMSCs achieved a greater recovery of brain function than hUC-MSCs and PBS in rats with HIE. CM derived from hPSC-EMSCs had neuroprotective and neurorestorative effects in vitro through anti-apoptotic and neurite outgrowth- and neurogenesis-promoting effects. Direct comparisons between hPSC-EMSCs and hUC-MSCs revealed the significant enrichment of a group of secretory factors in hPSC-EMSCs, including nerve growth factor (NGF), platelet-derived growth factor-AA and transforming growth factor-β2, which are involved in neurogenesis, synaptic transmission and neurotransmitter transport, respectively. Mechanistically, the CM derived from hPSC-EMSCs was found to potentiate NGF-induced neurite outgrowth and the neuronal differentiation of NPCs via the ERK/CREB pathway. Suppression of ERK or CREB abolished CM-potentiated neuritogenesis and neuronal differentiation. Finally, intranasal delivery of the CM derived from hPSC-EMSCs significantly reduced brain lesion size, promoted endogenous neurogenesis, mitigated inflammatory responses and improved functional recovery in rats with HIE.Conclusion: hPSC-EMSCs promote functional recovery after HI through multifaceted neuromodulatory activities via paracrine/trophic mechanisms. We propose the use of hPSC-EMSCs for the treatment of HIE, as they offer an excellent unlimited cellular source of MSCs.

Influence of microchannel geometry on device performance and electrophysiological recording fidelity during long-term studies of connected neural populations.

Compartmentalized microfluidic neural cell culture platforms, which physically separate axons from the neural soma using a series of microchannels, have been used for studying a wide range of pathological conditions and basic neuroscience questions. While each study has different experimental needs, the fundamental design of these devices has largely remained unchanged and a systematic study to establish long-term neural cultures in this format is lacking. Here, we investigate the influence of microchannel geometry and cell seeding density on device performance particularly in the context of long-term studies of synaptically-connected, yet fluidically-isolated neural populations of neurons and glia. Of the different experimental parameters, the microchannel height was the principal determinant of device performance, where the other parameters offer additional degrees of freedom in customizing such devices for specific applications. We condense the effects of these parameters into design rules and demonstrate their utility in engineering a microfluidic neural culture platform with integrated microelectrode arrays. The engineered device successfully recorded from primary rat cortical cells for 59 days in vitro with more than on order of magnitude enhancement in signal-to-noise ratio in the microchannels.

Engineering 3D Cortical Spheroids for an In Vitro Ischemic Stroke Model.

Three-dimensional (3D) spheroids composed of brain cells have shown great potential to mimic the pathophysiology of the brain. However, a 3D spheroidal brain-disease model for cerebral ischemia has not been reported. This study investigated an ultralow attachment (ULA) surface-mediated formation of 3D cortical spheroids using primary rat cortical cells to recapitulate the cerebral ischemic responses in stroke by oxygen-glucose deprivation-reoxygenation (OGD-R) treatment. Comparison between two-dimensional (2D) and 3D cell culture models confirmed the better performance of the 3D cortical spheroids as normal brain models. The cortical cells cultured in 3D maintained their healthy physiological morphology of a less activated state and suppressed mRNA expressions of pathological stroke markers, S100B, IL-1β, and MBP, selected based on in vivo stroke model. Interestingly, the spheroids formed on the ULA surface exhibited striking aggregation dynamics involving active cell-substrate interactions, whereas those formed on the agarose surface aggregated passively by the convective flow of the media. Accordingly, ULA spheroids manifested a layered arrangement of neurons and astrocytes with higher expressions of integrin β1, integrin α5, N-cadherin, and fibronectin than the agarose spheroids. OGD-R-induced stroke model of the ULA spheroids successfully mimicked the ischemic response as evidenced by the upregulated mRNA expressions of the key markers for stroke, S100B, IL-1β, and MBP. Our study suggested that structurally and functionally distinct cortical spheroids could be generated by simply tuning the cell-substrate binding activities during dynamic spheroidal formation, which should be an essential factor to consider in establishing a brain-disease model.

Culture of Rat Primary Cortical Cells for Microelectrode Array (MEA) Recordings to Screen for Acute and Developmental Neurotoxicity.

Neurotoxicity testing of chemicals, drug candidates, and environmental pollutants still relies on extensive in vivo studies that are very costly, time-consuming, and ethically debated due to the large number of animals typically used. Currently, rat primary cortical cultures are widely used for in vitro neurotoxicity studies, as they closely resemble the in vitro brain with respect to the diversity of cell types, their physiological functions, and the pathological processes that they undergo. Common in vitro assays for neurotoxicity screening often focus on very target-specific endpoints such as morphological, biochemical, or electrophysiological changes, and such narrow focus can hamper translation and interpretation. Microelectrode array (MEA) recordings provide a non-invasive platform for extracellular recording of electrical activity of cultured neuronal cells, thereby enabling the evaluation of changes in neuronal (network) function as a sensitive and integrated endpoint for neurotoxicity screening. Here, we describe an in vitro approach for assessing changes in neuronal network function as a measure for neurotoxicity, using rat primary cortical cultures grown on MEAs. We provide a detailed protocol for the culture of rat primary cortical cells, and describe several experimental procedures to address acute, subchronic, and chronic exposure scenarios. We additionally describe the steps for processing and analyzing MEA and cell viability data. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and culture of rat primary cortical cells on 48-well MEA plates Support Protocol 1: Pretreatment and washing of 48-well MEA plates before first use or for re-use Support Protocol 2: Coating of 48-well MEA plates with 0.1% PEI solution Basic Protocol 2: MEA measurements during acute exposure Alternate Protocol 1: MEA measurements during subchronic exposure Alternate Protocol 2: MEA measurements during chronic exposure Support Protocol 3: Determination of cell viability after MEA experiments Basic Protocol 3: MEA data processing Basic Protocol 4: Analyzing MEA experiments after acute and subchronic exposure Alternate Protocol 3: Analyzing MEA experiments after chronic exposure.

IL-2/IL-2R Antibody Complex Enhances Treg-Induced Neuroprotection by Dampening TNF-\u03b1 Inflammation in an In Vitro Stroke Model

The present in vitro study showed that IL-2/IL-2R antibody complex facilitates Treg-induced neuroprotection in the oxygen glucose deprivation/reoxygenation (OGD/R) model of stroke. First, we examined the role of IL-2/IL-2R-treated Tregs in OGD/R-exposed rat primary cortical cells (PCCs), which represent the cell type of the ischemic gray matter in the stroke brain. Here, OGD/R induced cell death, which was attenuated by Tregs and more robustly by IL-2/IL-2R-treated Tregs, but not by IL-2/IL-2R treatment alone. Second, we next assessed IL-2/IL-2R effects in OGD/R-exposed human oligodendrocyte progenitor cells (OPCs), which correspond to the white matter injury after stroke. Results revealed that a similar pattern neuroprotection as seen in the gray matter, in that OGD/R triggered cell death, which was ameliorated by Tregs and more effectively by IL-2/IL-2R-treated Tregs, but IL-2/IL-2R treatment alone was not therapeutic. Third, as we begin to understand the mechanism underlying IL-2/IL-2R engagement of Tregs, we investigated the inflammatory response in OGD/R-exposed human neural progenitor cells (NPCs), which recapitulate both ischemic gray and white matter damage in stroke. Similar to PCCs and OPCs, OGD/R produced cell death and was blocked by Tregs and more efficiently by IL-2/IL-2R-treated Tregs, whereas IL-2/IL-2R treatment alone did not alter the ischemic insult. Moreover, the inflammatory marker, TNF-α, was upregulated after OGD/R, dampened by both Tregs and more efficiently by IL-2/IL-2R-treated Tregs but more pronounced in the latter, and not affected by IL-2/IL-2R treatment alone, suggesting IL-2/IL-2R-Treg-mediated modulation of inflammatory response in stroke. Altogether, these observations support the use of IL-2/IL-2R treatment in enhancing the anti-inflammatory effects of Tregs in stroke.

Structurally different anabolic androgenic steroids reduce neurite outgrowth and neuronal viability in primary rat cortical cell cultures

The illicit use of anabolic androgenic steroids (AAS) among adolescents and young adults is a major concern due to the unknown and unpredictable impact of AAS on the developing brain and the consequences of this on mental health, cognitive function and behaviour. The present study aimed to investigate the effects of supra-physiological doses of four structurally different AAS (testosterone, nandrolone, stanozolol and trenbolone) on neurite development and cell viability using an in vitro model of immature primary rat cortical cell cultures. A high-throughput screening image-based approach, measuring the neurite length and number of neurons, was used for the analysis of neurite outgrowth. In addition, cell viability and expression of the Tubb3 gene (encoding the protein beta-III tubulin) were investigated. Testosterone, nandrolone, and trenbolone elicited adverse effects on neurite outgrowth as deduced from an observed reduced neurite length per neuron. Trenbolone was the only AAS that reduced the cell viability as indicated by a decreased number of neurons and declined mitochondrial function. Moreover, trenbolone downregulated the Tubb3 mRNA expression. The adverse impact on neurite development was neither inhibited nor supressed by the selective androgen receptor (AR) antagonist, flutamide, suggesting that the observed effects result from another mechanism or mechanisms of action that are operating apart from AR activation. The results demonstrate a possible AAS-induced detrimental effect on neuronal development and regenerative functions. An impact on these events, that are essential mechanisms for maintaining normal brain function, could possibly contribute to behavioural alterations seen in AAS users.

Different effects of metformin and phenformin on hypoxia-induced Ca2+ fluxes in cultured primary neurons

Recent evidence suggests that metformin and phenformin may exert beneficial effects against neuronal injury in the ischemic brain, however, the difference of action between these two drugs and the molecular mechanism of such protection is not clear. In this study, we investigated whether mild hypoxia-affected neurons exhibit changes in cytosolic calcium handling and whether metformin and phenformin exert any effect on calcium homeostasis in hypoxia-affected neurons. Cultured primary rat cortical cells were stained with calcium sensitive dye Oregon Green 488 BAPTA-1 AM and spontaneous calcium dependent changes of fluorescence were recorded. Using obtained fluorescence traces we estimated changes in relative amplitude of recorded spontaneous signals, changes in frequency of spontaneous activity, and changes in decay of fluorescence traces. We found that hypoxia caused reduction of the relative signal amplitude, increased the spontaneous activity, and slowed the decay of calcium concentration. After pre-treatment of cells with 0.1–0.5 mM metformin, the relative signal amplitude increased and the frequency of spontaneous signals decreased in hypoxia-affected neurons. However, pre-treatment with 1–25 µM phenformin neither increased the relative signal amplitude nor reduced the frequency of spontaneous signals. The decay of fluorescence traces became faster after application of metformin or phenformin comparing to neurons under hypoxic conditions. These results suggest different action of metformin and phenformin in improvement of Ca2+ homeostasis in hypoxia-affected neurons, which may have different effects on neuronal survival and functions after hypoxia/ischemia.

RNA Binding Protein Motif 3 Inhibits Oxygen-Glucose Deprivation/Reoxygenation-Induced Apoptosis Through Promoting Stress Granules Formation in PC12 Cells and Rat Primary Cortical Neurons.

As a sensitive cold-shock protein, RNA binding protein motif 3 (RBM3) exhibits a neuroprotective function in the condition of brain injury. However, how RBM3 is involved in acute ischemic stroke by affecting stress granules (SGs) remains unclear. Here, we established an oxygen-glucose deprivation/reperfusion (OGD/R) model in rat primary cortical neurons and PC12 cells to explore the potential mechanism between RBM3 and SG formation in acute ischemic/reperfusion (I/R) condition. The immunofluorescence results showed that the SG formation significantly decreased in rat primary cortical neurons and PC12 cells during the reperfusion period after 6 h of OGD stimulation. The western blot results, flow cytometry analysis, and cell viability assessment showed that the RBM3 expression and ratio of cell viability significantly decreased, while the rate of apoptosis increased in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Co-immunoprecipitation (Co-IP) and immunofluorescence indicated that RBM3 and GTPase-activating protein-binding protein 1 (G3BP1) colocalized cytoplasm of PC12 cells after 6 h of OGD stimulation when the SGs formation reached the highest level. Besides, overexpression and knockdown of the RBM3 were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. The results of overexpression and knockdown of RBM3 gene illustrated the pivotal role of RBM3 in affecting SG formation and apoptosis level in OGD-treated PC12 cells. In conclusion, RBM3 could combine with G3BP1 resulted in increasing stress granules generation in rat primary cortical neurons and PC12 cells after 6 h of oxygen-glucose deprivation (OGD) injury, which ultimately reduced the apoptosis in OGD-induced cells. Our study may enable a new promising target for alleviating ischemia-reperfusion injury in cells.

Screening of tau protein kinase inhibitors in a tauopathy-relevant cell-based model of tau hyperphosphorylation and oligomerization

Tauopathies are a class of neurodegenerative disorders characterized by abnormal deposition of post-translationally modified tau protein in the human brain. Tauopathies are associated with Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE), and other diseases. Hyperphosphorylation increases tau tendency to aggregate and form neurofibrillary tangles (NFT), a pathological hallmark of AD. In this study, okadaic acid (OA, 100 nM), a protein phosphatase 1/2A inhibitor, was treated for 24h in mouse neuroblastoma (N2a) and differentiated rat primary neuronal cortical cell cultures (CTX) to induce tau-hyperphosphorylation and oligomerization as a cell-based tauopathy model. Following the treatments, the effectiveness of different kinase inhibitors was assessed using the tauopathy-relevant tau antibodies through tau-immunoblotting, including the sites: pSer202/pThr205 (AT8), pThr181 (AT270), pSer202 (CP13), pSer396/pSer404 (PHF-1), and pThr231 (RZ3). OA-treated samples induced tau phosphorylation and oligomerization at all tested epitopes, forming a monomeric band (46–67 kDa) and oligomeric bands (170 kDa and 240 kDa). We found that TBB (a casein kinase II inhibitor), AR and LiCl (GSK-3 inhibitors), cyclosporin A (calcineurin inhibitor), and Saracatinib (Fyn kinase inhibitor) caused robust inhibition of OA-induced monomeric and oligomeric p-tau in both N2a and CTX culture. Additionally, a cyclin-dependent kinase 5 inhibitor (Roscovitine) and a calcium chelator (EGTA) showed contrasting results between the two neuronal cultures. This study provides a comprehensive view of potential drug candidates (TBB, CsA, AR, and Saracatinib), and their efficacy against tau hyperphosphorylation and oligomerization processes. These findings warrant further experimentation, possibly including animal models of tauopathies, which may provide a putative Neurotherapy for AD, CTE, and other forms of tauopathy-induced neurodegenerative diseases.

Nanocomposite-Coated Silk-Based Artificial Conduits: The Influence of Structures on Regeneration of the Peripheral Nerve.

In this study, silk filaments are coated with different concentrations (5, 7.5, and 10% w/w) of carbon nanofibers (CNFs) dispersed in poly-ε-caprolactone. The nanocomposite-coated silk filaments are subjected to knitting, braiding, and twisting. The tubular structures are covered with a silk fibroin/polyvinyl film for the nerve conduit application. Physical characterization of the developed nerve conduits demonstrates suitable mechanical properties comparable to native nerve tissue. Cell proliferation is confirmed through in vitro cell culture studies using Neuro 2a and rat primary cortical neural progenitor cells, which show that the proliferation happens along the interconnected macrochannels of the internal structure of the nerve conduit. The knitted structure presents better biological properties than the nerve conduits with other internal structures. The in vivo sciatic nerve implantation is performed in a rabbit model using the best conduit, i.e., 10% CNF-based nanocomposite-coated silk with a knitted inner structure without any biomolecules or tube filling gels. Regeneration of a 2 cm gap excised sciatic nerve is investigated by immunohistochemistry and histology of implanted nerve conduits removed after 30 days. Results suggest that the CNF-based conducting nanocomposite coating in this well-defined architecture of the conduit helps in signal transmission and neural growth during the regeneration of the transected nerve.

Idebenone Has Distinct Effects on Mitochondrial Respiration in Cortical Astrocytes Compared to Cortical Neurons Due to Differential NQO1 Activity.

Idebenone is a synthetic quinone that on reduction in cells can bypass mitochondrial Complex I defects by donating electrons to Complex III. The drug is used clinically to treat the Complex I disease Leber's hereditary optic neuropathy (LHON), but has been less successful in clinical trials for other neurodegenerative diseases. NAD(P)H:quinone oxidoreductase 1 (NQO1) appears to be the main intracellular enzyme catalyzing idebenone reduction. However, NQO1 is not universally expressed by cells of the brain. Using primary rat cortical cells pooled from both sexes, we tested the hypotheses that the level of endogenous NQO1 activity limits the ability of neurons, but not astrocytes, to use idebenone as an electron donor to support mitochondrial respiration. We then tested the prediction that NQO1 induction by pharmacological activation of the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) enables idebenone to bypass Complex I in cells with poor NQO1 expression. We found that idebenone stimulated respiration by astrocytes but reduced the respiratory capacity of neurons. Importantly, idebenone supported mitochondrial oxygen consumption in the presence of a Complex I inhibitor in astrocytes but not neurons, and this ability was reversed by inhibiting NQO1. Conversely, recombinant NQO1 delivery to neurons prevented respiratory impairment and conferred Complex I bypass activity. Nrf2 activators failed to increase NQO1 in neurons, but carnosic acid induced NQO1 in COS-7 cells that expressed little endogenous enzyme. Carnosic acid-idebenone combination treatment promoted NQO1-dependent Complex I bypass activity in these cells. Thus, combination drug strategies targeting NQO1 may promote the repurposing of idebenone for additional disorders.SIGNIFICANCE STATEMENT Idebenone is used clinically to treat loss of visual acuity in Leber's hereditary optic neuropathy. Clinical trials for several additional diseases have failed. This study demonstrates a fundamental difference in the way idebenone affects mitochondrial respiration in cortical neurons compared with cortical astrocytes. Cortical neurons are unable to use idebenone as a direct mitochondrial electron donor due to NQO1 deficiency. Our results suggest that idebenone behaves as an NQO1-dependent prodrug, raising the possibility that lack of neuronal NQO1 activity has contributed to the limited efficacy of idebenone in neurodegenerative disease treatment. Combination therapy with drugs able to safely induce NQO1 in neurons, as well as other brain cell types, may be able to unlock the neuroprotective therapeutic potential of idebenone or related quinones.