Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

The pathological basis of neonatal hypoxia–ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI‐induced neuronal cell death. The p38 mitogen‐activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen/glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase‐3 and the appearance of apoptotic neuronal cells. Pre‐treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I‐1‐driven adeno‐associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD‐mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD.     

LncRNA SNHG12 ameliorates brain microvascular endothelial cell injury by targeting miR-199a

Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation(OGD/R) insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.     

Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.     

P2X7 receptor blockade prevents ATP excitotoxicity in neurons and reduces brain damage after ischemia

Overactivation of subtype P2X7 receptors can induce excitotoxic neuronal death by calcium (Ca(2+)) overload. In this study, we characterize the functional properties of P2X7 receptors using electrophysiology and Ca(2+) monitoring in primary cortical neuron cultures and in brain slices. Both electrical responses and Ca(2+) influx induced by ATP and benzoyl-ATP were reduced by Brilliant Blue G (BBG) at concentrations which specifically inhibit P2X7 receptors. In turn, oxygen-glucose deprivation (OGD) caused neuronal death that was reduced with BBG application. OGD in neuron cultures and brain slices generated an inward current, which was delayed and reduced by BBG. To assess the relevance of these in vitro findings, we used middle cerebral artery occlusion in rats as a model of transient focal cerebral ischemia to study the neuroprotective effect of BBG in vivo. Treatment with BBG (twice per day, 30 mg/kg) produced a 60% reduction in the extent of brain damage compared to treatment with vehicle alone. These results show that P2X7 purinergic receptors mediate tissue damage after OGD in neurons and following transient brain ischemia. Therefore, these receptors are a relevant molecular target for the development of new treatments to attenuate brain damage following stroke.     

Evidence for the involvement of Par-4 in ischemic neuron cell death.

After a stroke many neurons in the ischemic brain tissue die by a process called apoptosis, a form of cell death that may be preventable. The specific molecular cascades that mediate ischemic neuronal death are not well understood. The authors recently identified prostate apoptosis response-4 (Par-4) as a protein that participates in the death of cultured hippocampal neurons induced by trophic factor withdrawal and exposure to glutamate. Here, the authors show that Par-4 levels increase in vulnerable populations of hippocampal and striatal neurons in rats after transient forebrain ischemia; Par-4 levels increased within 6 hours of reperfusion and remained elevated in neurons undergoing apoptosis 3 days later. After transient focal ischemia in mice, Par-4 levels were increased 6 to 12 hours after reperfusion in the infarcted cortex and the striatum, and activation of caspase-8 occurred with a similar time course. Par-4 immunoreactivity was localized predominantly in cortical neurons at the border of the infarct area. A Par-4 antisense oligonucleotide protected cultured hippocampal neurons against apoptosis induced by chemical hypoxia and significantly reduced focal ischemic damage in mice. The current data suggest that early up-regulation of Par-4 plays a pivotal role in ischemic neuronal death in animal models of stroke and cardiac arrest.     

Excessive Autophagy Contributes to Neuron Death in Cerebral Ischemia

Aims: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. Methods: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo. Results: In both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3–7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. Conclusion: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.     

Inhibition of calcium/calmodulin‐dependent protein kinase kinase (CaMKK) exacerbates impairment of endothelial cell and blood–brain barrier after stroke

Brain microvascular endothelial cells play an essential role in maintaining blood–brain barrier (BBB) integrity, and disruption of the BBB aggravates the ischemic injury. CaMKK (α and β) is a major kinase activated by elevated intracellular calcium. Previously, we demonstrated that inhibition of CaMKK exacerbated outcomes, conversely, overexpression reduced brain injury after stroke in mice. Interestingly, CaMKK has been shown to activate a key endothelial protector, sirtuin 1 (SIRT1). We hypothesized that CaMKK protects brain endothelial cells via SIRT1 activation after stroke. In this study, Oxygen‐Glucose Deprivation (OGD) was performed in human brain microvascular endothelial cells. Stroke was induced by middle cerebral artery occlusion (MCAO) in male mice. Knockdown of CaMKK β using siRNA increased cell death following OGD. Inhibition of CaMKK β by STO‐609 significantly and selectively down‐regulated levels of phosphorylated SIRT1 after OGD. Changes in the downstream targets of SIRT1 were observed following STO‐609 treatment. The effect of STO‐609 on cell viability after OGD was absent, when SIRT1 was concurrently inhibited. We also demonstrated that STO‐609 increased endothelial expression of the pro‐inflammatory proteins ICAM‐1 and VCAM‐1 and inhibition of CaMKK exacerbated OGD‐induced leukocyte‐endothelial adhesion. Finally, intracerebroventricular injection of STO‐609 exacerbated endothelial apoptosis and reduced BBB integrity after 24‐hr reperfusion following MCAO in vivo. Collectively, these results demonstrated that CaMKK inhibition reduced endothelial cell viability, exacerbated inflammatory responses and aggravated BBB impairment after ischemia. CaMKK activation may attenuate ischemic brain injury via protection of the microvascular system and a reduction in the infiltration of pro‐inflammatory factors.     

Mesenchymal stem cells attenuate MRI‐identifiable injury,protect white matter,and improve long‐term functional outcomes after neonatal focal stroke in rats

Cell therapy has emerged as a potential treatment for many neurodegenerative diseases including stroke and neonatal ischemic brain injury. Delayed intranasal administration of mesenchymal stem cells (MSCs) after experimental hypoxia‐ischemia and after a transient middle cerebral artery occlusion (tMCAO) in neonatal rats has shown improvement in long‐term functional outcomes, but the effects of MSCs on white matter injury (WMI) are insufficiently understood. In this study we used longitudinal T2‐weighted (T2W) and diffusion tensor magnetic resonance imaging (MRI) to characterize chronic injury after tMCAO induced in postnatal day 10 (P10) rats and examined the effects of delayed MSC administration on WMI, axonal coverage, and long‐term somatosensory function. We show unilateral injury‐ and region‐dependent changes in diffusion fraction anisotropy 1 and 2 weeks after tMCAO that correspond to accumulation of degraded myelin basic protein, astrocytosis, and decreased axonal coverage. With the use of stringent T2W‐based injury criteria at 72 hr after tMCAO to randomize neonatal rats to receive intranasal MSCs or vehicle, we show that a single MSC administration attenuates WMI and enhances somatosensory function 28 days after stroke. A positive correlation was found between MSC‐enhanced white matter integrity and functional performance in injured neonatal rats. Collectively, these data indicate that the damage induced by tMCAO progresses over time and is halted by administration of MSCs. © 2016 Wiley Periodicals, Inc.     

Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1alpha-linked induction of P450 2C11 epoxygenase in astrocytes.

The brain's adaptive response to ischemic preconditioning (IPC) is mediated in part via hypoxia inducible factor (HIF)-responsive genes. We previously showed that IPC induces cytochrome P450 2C11 expression in the brain, associated with protection from stroke. Cytochrome P450 2C11 is an arachidonic acid (AA) epoxygenase expressed in astrocytes, which metabolizes AA to epoxyeicosatrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1alpha, and that the P450 epoxygenase pathway contributes to enhanced astrocyte tolerance to ischemia-like injury induced by oxygen-glucose deprivation (OGD). Primary cultured astrocytes were incubated under normoxic or hypoxic conditions for 1, 3, 6, 24, or 48 h, and protein levels of P450 2C11 and HIF-1alpha were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1alpha to 2C11 promoter was evaluated using electrophoretic mobility shift assay (EMSA) with 2C11 promoter DNA containing putative HIF-binding sites. Levels of 2C11 mRNA and protein were significantly increased starting at 3 and 6 h of hypoxia, respectively. The increase in 2C11 expression was preceded by an increase in HIF-1alpha protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1alpha in nuclear extracts from astrocytes. HPC and EETs reduced astrocyte cell death, and P450 epoxygenase inhibition prevented protection by HPC. We conclude that HPC induces tolerance in astrocytes, at least in part, via HIF-1alpha-linked upregulation of P450 2C11.     

Neuroprotective effects of extracellular DJ‐1 on reperfusion injury in SH‐SY5Y cells

Using an in vitro model of ischemic stroke we treated differentiated SH‐SY5Y cells to oxygen‐glucose deprivation (OGD) followed by a reperfusion period where normal growth conditions were restored. Cells undergoing OGD exhibited significant cell death as measure by propidium iodide staining. However, cells treated with exogenous extracellular DJ‐1 during reperfusion exhibited significant rescue from OGD‐induced cell death.     

17β‐estradiol protects against hypoxic/ischemic white matter damage in the neonatal rat brain

Developing oligodendrocytes (pre‐OLs) are highly vulnerable to hypoxic‐ischemic injury and associated excitotoxicity and oxidative stress. 17β‐Estradiol plays an important role in the development and function of the CNS and is neuroprotective. The sudden drop in circulating estrogen after birth may enhance the susceptibility of developing OLs to injury. Estrogen receptor (ER)–α and ER‐β are both expressed in OLs. We examined the effect of 17β‐estradiol on oxygen‐glucose deprivation and oxidative stress–induced cell death in rat pre‐OLs in vitro and on hypoxic‐ischemic brain injury in vivo. Pre‐OLs in culture were subjected to oxygen‐glucose deprivation (OGD) or glutathione depletion in the presence or absence of 17β‐estradiol. LDH release, the Alamar blue assay, and phase‐contrast microscopy were used to assess cell viability. Hypoxic‐ischemic injury was generated in 6‐day‐old rats (P6) by unilateral carotid ligation and hypoxia (6% O₂ for 1 hr). Rat pups received one intraperitoneal injection of 300 or 600 μg/kg 17β‐estradiol or vehicle 12 hr prior to the surgical procedure. Injury was assessed by myelin basic protein (MBP) immunocytochemistry at P10. 17β‐Estradiol produced significant protection against OGD‐induced cell death in primary OLs (EC₅₀ = 1.3 ± 0.46 × 10^?9 M) and against oxidative stress. Moreover, 17β‐estradiol attenuated the loss of MBP labeling in P10 pups ipsilateral to the carotid ligation. These results suggest a potential role for estrogens in attenuation of hypoxic‐ischemic and oxidative injury to developing OLs and in the prevention of periventricular leukomalacia. © 2009 Wiley‐Liss, Inc.     

Hypoxia-controlled matrix metalloproteinase-9 hyperexpression promotes behavioral recovery after ischemia

Matrix metalloproteinase-9(MMP-9) plays a beneficial role in the sub-acute phase after ischemic stroke.However,unrestrained MMP-9 may disrupt the blood-brain barrier(BBB),which has limited its use for the treatment of brain ischemia.In the present study,we constructed lentivirus mediated hypoxiacontrolled MMP-9 expression and explored its role after stroke.Hypoxia response element(HRE)was used to confine MMP-9 expression only to the hypoxic region of mouse brain after 120-min transient middle cerebral artery occlusion.Lentiviruses were injected into the peri-infarct area on day 7 after transient ischemia.We found hyperexpression of exogenous HRE-MMP-9 under the control of hypoxia,and its expression was mainly located in neurons and astrocytes without aggravation of BBB damage compared to the CMV group.Furthermore,mice in the HRE-MMP-9 group showed the best behavioral recovery compared with the normal saline,GFP,and SB-3CT groups.Therefore,hypoxia-controlled MMP-9 hyperexpression during the sub-acute phase of ischemia may provide a novel promising approach of gene therapy for stroke.     

Bcl-2 family members make different contributions to cell death in hypoxia and/or hyperoxia in rat cerebral cortex

Hypoxic brain injury during fetal or neonatal development leads to damaged immature neurons and can result in cognitive or behavioral dysfunction. Hyperoxia therapy (treatment with oxygen) is commonly applied to infants with signs of perinatal hypoxia-anoxia. Both hypoxia and hyperoxia have been shown to result in apoptosis in the brains of rats in several animal models. One determinant of cellular commitment to cell death is the differential expression of the Bcl-2 family of proteins in response to trauma. Here, we characterize cell death and the expression of Bcl-2 homologous proteins in 7-day-old neonatal rat cerebral cortex after hypoxia (5% O(2) for 40 min) and/or hyperoxia (>95% O(2) for 2 h after hypoxia). The expression of Bcl-2 and Bcl-X(L), two anti-apoptotic proteins, decreased at 24 h after hypoxia. Bcl-X(L) increased after either hyperoxia or hypoxia+hyperoxia. We did not detect significant changes in the cytoplasmic levels of pro-apoptotic protein Bax after any of these three treatments. Using cell death ELISA and DNA FragEL assays, we observed increased cell death at 24h after hypoxia, hyperoxia or hypoxia+hyperoxia treatments. At 24 h after either hypoxia, hyperoxia or hypoxia+hyperoxia, caspase 3 activity also increased significantly. Our results suggest that both hypoxia and hyperoxia alone can induce cell death. The Bcl-2 --> cytochrome c --> caspase 3 pathway played a role in hypoxia-induced cell death, while other pathways may be involved in hyperoxia-induced cell death.     

TNP-ATP is Beneficial for Treatment of Neonatal Hypoxia-Induced Hypomyelination and Cognitive Decline

Our previous study together with other investigations have reported that neonatal hypoxia or ischemia induces long-term cognitive impairment, at least in part through brain inflammation and hypomyelination. However, the detailed mechanisms are not fully understood.Here, we used a rodent model of neonatal hypoxia by subjecting postnatal day 0(P0) rat pups to systemic hypoxia(3.5 h). We found that neonatal hypoxia increased the glutamate content and initiated inflammatory responses at 4 h and 1 day after hypoxia, caused hypomyelination in the corpus callosum, and impaired hippocampus-dependent learning and memory when assessed 30–60 days after hypoxia. Interestingly, much of the hypoxia-induced brain damage was ameliorated by treatment with the ATP analogue 20,30-0-(2,4,6-trinitrophenyl)-adenosine 50-triphosphate(TNP-ATP; blocks all ionotropic P2X1-7 receptors),whereas treatment with pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid(PPADS; inhibits P2X1-3 and P2X5-7 receptors) was less neuroprotective. Our data indicated that activation of ionotropic ATP receptors might be partially, if not fully, involved in glutamate deregulation,neuroinflammation, hypomyelination, and cognitive dysfunction after neonatal hypoxia.     

Role of P450 aromatase in sex-specific astrocytic cell death.

Female animals are protected from ischemic brain damage relative to age-matched males, in part through protection provided by endogenous estradiol. In brain, estradiol is produced from testosterone by cytochrome P450 aromatase (cyp 19), a steroid synthetic enzyme present in astrocytes. We tested the hypothesis that astrocytes derived from neonatal female rat brain are less susceptible than male cells to oxygen-glucose deprivation (OGD), and that this endogenous protection is associated with enhanced aromatase activity. Primary cultured cortical astrocytes were prepared from male and female rat pups separately and grown to confluence in estrogen-free media. Cell death in response to OGD, alone or in combination with hydrogen peroxide, lipopolysaccharides, interleukin-1beta, tissue necrosis factor-alpha, or nitric oxide (NO) donor diethylenetriamine/nitric oxide adduct (DETA/NO) was quantified as the ratio of propidium iodide to calcein AM-positive cells. Aromatase activity and cyp19 mRNA and protein levels were measured in cultures from each sex. Female astrocytes are more resistant to OGD and oxidant cell death induced by H(2)O(2) , but sustain greater cell death when inflammatory mediators are combined with OGD compared with OGD alone. Media transfer from female to male cells conferred protection against OGD-induced cell death. Aromatase activity and expression is greater in female than in male astrocytes. The aromatase inhibitor, Arimidex (100 nmol/L), abolishes sex differences in OGD-induced cell death, whereas treatment with 17beta-estradiol (10 nmol/L) protects cells of either sex. We conclude that astrocytes isolated from neonatal cortex exhibit marked sex differences in sensitivity to OGD, in part because of enhanced aromatization and estradiol formation in female cells.     

Activin is a neuronal survival factor that is rapidly increased after transient cerebral ischemia and hypoxia in mice.

One approach for developing targeted stroke therapies is to identify the neuronal protective and destructive signaling pathways and gene expression that follow ischemic insult. In some neural injury models, the transforming growth factor-beta family member activin can provide neuroprotective effects in vivo and promote neuronal survival. This study tests if activin supports cortical neurons after ischemic challenge in vitro and if signals after cerebral ischemia involve activin in vivo. In a defined cell culture model that uses hydrogen peroxide (H(2)O(2))-free radical stress, activin addition maintained neuronal survival. H(2)O(2) treatment increased activin mRNA twofold in surviving cortical neurons, and inhibition of activin with neutralizing antibodies caused neuronal death. These data identify activin gene changes as a rapid response to oxidative stress, and indicate that endogenous activin acts as a protective factor for cortical neurons in vitro. Similarly, after transient focal cerebral ischemia in adult mice, activin mRNA increased at 1 and 4 h ipsilateral to the infarct but returned to control values at 24 h after reperfusion. Intracellular activated smad signals were detected in neurons adjacent to the infarct. Activin was also increased after 2 h of 11% hypoxia. Activin mRNA increased at 1 h but not 4 or 24 h after hypoxia, similar to the time course of erythropoietin and vascular endothelial growth factor induction. These findings identify activin as an early-regulated gene response to transient ischemia and hypoxia, and its function in cortical neuron survival during oxidative challenge provides a basis to test activin as a potential therapeutic in stroke injury.     

Resident microglia,rather than blood‐derived macrophages,contribute to the earlier and more pronounced inflammatory reaction in the immature compared with the adult hippocampus after hypoxia‐ischemia

The mechanisms of neuronal injury after hypoxia–ischemia (HI) are different in the immature and the adult brain, but microglia activation has not been compared. The purpose of this study was to phenotype resident microglia and blood‐derived macrophages in the hippocampus after HI in neonatal (postnatal day 9, P9) or adult (3 months of age, 3mo) mice. Unilateral brain injury after HI was induced in Cx3cr1^GFP/+Ccr2^RFP/+ male mice on P9 (n = 34) or at 3mo (n = 53) using the Vannucci model. Resident microglia (Cx3cr1‐GFP+) proliferated and were activated earlier after HI in the P9 (1–3 days) than that in the 3mo hippocampus, but remained longer in the adult brain (3–7 days). Blood‐derived macrophages (Ccr2‐RFP+) peaked 3 days after HI in both immature (P9) and adult (3mo) hippocampi but were twice as frequent in adult brains, 41% vs. 21% of all microglia/macrophages. CCL2 expression was three times higher in the P9 hippocampi, indicating that the proinflammatory response was more pronounced in the immature brain after HI. This corresponded well with the higher numbers of galectin‐3‐positive resident microglia in the P9 hippocampi, but did not correlate with CD16/32‐ or CD206‐positive resident microglia or blood‐derived macrophages. In conclusion, resident microglia, rather than infiltrating blood‐derived macrophages, proliferate and are activated earlier in the immature than in the adult brain, but remain increased longer in the adult brain. The inflammatory response is more pronounced in the immature brain, and this correlate well with galectin‐3 expression in resident microglia. GLIA 2015;63:2220–2230     

Preservation of neuronal functions by exosomes derived from different human neural cell types under ischemic conditions

Stem cell‐based therapies have been reported in protecting cerebral infarction‐induced neuronal dysfunction and death. However, most studies used rat/mouse neuron as model cell when treated with stem cell or exosomes. Whether these findings can be translated from rodent to humans has been in doubt. Here, we used human embryonic stem cell‐derived neurons to detect the protective potential of exosomes against ischemia. Neurons were treated with in vitro oxygen–glucose deprivation (OGD) for 1 h. For treatment group, different exosomes were derived from neuron, embryonic stem cell, neural progenitor cell and astrocyte differentiated from H9 human embryonic stem cell and added to culture medium 30 min after OGD (100 μg/mL). Western blotting was performed 12 h after OGD, while cell counting and electrophysiological recording were performed 48 h after OGD. We found that these exosomes attenuated OGD‐induced neuronal death, Mammalian target of rapamycin (mTOR), pro‐inflammatory and apoptotic signaling pathway changes, as well as basal spontaneous synaptic transmission inhibition in varying degrees. The results implicate the protective effect of exosomes on OGD‐induced neuronal death and dysfunction in human embryonic stem cell‐derived neurons, potentially through their modulation on mTOR, pro‐inflammatory and apoptotic signaling pathways.