Primary Cortical Cells Research Articles

Neuroprotective effects of erythromycin on cerebral ischemia reperfusion-injury and cell viability after oxygen-glucose deprivation in cultured neuronal cells

This study aims to determine if erythromycin has neuroprotective effects against transient ischemia and oxygen-glucose deprivation (OGD) in cultured neuronal cells. Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 90min, followed by reperfusion. The animals received a subcutaneous single injection of erythromycin lactobionate (EM, 50mg/kg) or vehicle immediately after ischemia. Infarct volume, edema index, and neurological performance were evaluated at 24 and 72h after reperfusion. Immunohistochemical analyses for oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) were conducted in the cortex at 24h. Primary cortical neuronal cell cultures were prepared from the cerebral cortices of the animals and then subjected to OGD for 3h. Ten or 100μM EM was added before OGD to determine the effect of EM on cell viability after OGD. EM significantly reduced infarct volume (p<0.01) and edema volume (p<0.05) and improved neurological deficit scores (p<0.05) at 24 and 72h. EM significantly suppressed the accumulation of 4-HNE (p<0.01) and 8-OHdG (p<0.01) and markedly reduced Iba-1 (p<0.01) and TNF-α expression (p<0.01). Treatment with 100M EM in vitro significantly reduced cell death after OGD. EM reduces neuronal damage following cerebral ischemia and OGD and may have antioxidant and anti-inflammatory effects.

Phosphatidylserine and curcumin act synergistically to down-regulate release of interleukin-1β from lipopolysaccharide-stimulated cortical primary microglial cells.

Microglia, the brain's resident macrophages, contribute to immune surveillance and the response to disease and injury. These immune cells play a dual role in the nervous system, having both neurotoxic and neuroprotective effects. Activation of microglia results in the production of inflammatory molecules and neurotoxic factors that often cause or contribute to neurodegenerative diseases. Inhibition of neurotoxic microglia activation and consequent inflammatory processes may represent an important therapeutic target. Phosphatidylserine (PS), an aminophospholipid of plasma membranes, and curcumin, the yellow pigment isolated from the rhizome of the turmeric plant, have both been reported to suppress microglial activation by reducing pro-inflammatory mediator production and release. In this study we analyzed the effects of PS, curcumin, and their association on microglial activation induced by the bacterial toxin lipopolysaccharide. Primary rat cortical microglial cells were treated with increasing concentrations of PS-liposomes and curcumin, alone or in combination, and their effects on pro-inflammatory cytokine release from unstimulated and lipopolysaccharide-stimulated microglia were evaluated by enzyme-linked immunosorbent assay. Isobolographic analysis was performed to investigate the effect of PS-liposomes and curcumin combination. PS and curcumin inhibited the release of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α induced by lipopolysaccharide. Furthermore, PS and curcumin in combination exerted a synergistic effect in down-regulating IL-1β release. These results suggest that the association of PS with curcumin could be of potential therapeutic utility against diseases associated with microglial activation.

Upregulation of HIF-1α protein induces mitochondrial autophagy in primary cortical cell cultures through the inhibition of the mTOR pathway.

Cerebral ischemia/reperfusion (I/R) can induce neuronal death, particularly in the hippocampal formation (HF). Molecular genetic studies have suggested that the activities of the transcription factor, hypoxia-inducible factor-1α (HIF-1α), are closely linked to ischemia-induced neuronal death. However, the mechanisms through which HIF-1α functions remain poorly understood. In this study, primary cortical neurons were subjected to oxygen‑glucose deprivation (OGD) to establish a cell model of OGD/reperfusion (RP). HIF-1α mRNA and protein expression was measured by qRT-PCR and western blot analysis. Cell proliferation was detected by MTT assay. Flow cytometric analysis was used to detect cell apoptosis and changes in mitochondrial mass. The expression of LC3-Ⅰ and LC3-Ⅱ was examined by western blot analysis. We found that HIF-1α increased cell proliferation and decreased cell apoptosis in our cell model of OGD/RP using cultured neonatal rat cortical neurons. The overexpression of HIF-1α significantly induced changes in mitochondrial mass and mitochondrial autophagy in cortical neurons. Moreover, the inhibition of HIF-1α markedly suppressed cell proliferation and mitochondrial autophagy. We also demonstrated that the HIF-1α-induced mitochondrial autophagy was accompanied by the inhibition of the mTOR pathway. This study provides direct in vitro evidence that HIF-1α overexpression triggers mitochondrial autophagy, thereby increasing neuronal survival. Our results highlight a novel target molecule toward which anti-ischemic neuroprotective effects can be applied.

2-Cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against beta-amyloid-induced activation of the apoptotic cascade in cultured cortical neurons.

Aggregated β-amyloid, implicated in the pathogenesis of Alzheimer's disease (AD), induces neurotoxicity by evoking a cascade of oxidative damage-dependent apoptosis in neurons. We investigated the molecular mechanisms underlying the protective effect of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride (KHG26377) against the beta-amyloid (Aβ25-35)-induced primary cortical neuronal cell neurotoxicity. Treatment with KHG26377 attenuated the Aβ25-35-induced apoptosis by decreasing the Bax/Bcl-2 ratio and suppressing the activation of caspase-3. A marked increase in calcium influx and in the level of reactive oxygen species together with a decrease in glutathione levels was found after Aβ25-35 exposure; however, KHG26377 treatment reversed these changes in a concentration-dependent manner. In addition, KHG26377 significantly suppressed Aβ25-35-induced toxicity concomitant with a reduction in the activation of extracellular signal-regulated kinases 1 and 2 and nuclear factor kappa B. The KHG26377-induced protection of neuronal cells against Aβ toxicity was also mediated by suppressing the expression of glycogen synthase kinase-3β, increasing the levels of β-catenin, and reducing the levels of phosphorylated tau. Our findings suggest that KHG26377 may modulate the neurotoxic effects of β-amyloid and provide a rationale for treatment of AD.

Neuroprotective effects of diarylpropionitrile against β-amyloid peptide-induced neurotoxicity in rat cultured cortical neurons

Alzheimer's disease is a major cause of dementia in the elderly that involves a β-amyloid peptide (Aβ)-induced cascade of an increase in oxidative damage and inflammation. The present study demonstrated the neuroprotective effects of diarylpropionitrile (DPN), a non-steroidal estrogen receptor β selective ligand, against 10μM Aβ1–42-induced oxidative stress and inflammation in primary rat cortical cell culture. Pre-treatment with 1–100nM DPN significantly decreased neuronal cell death by increasing cell viability through a significant attenuation in the reactive oxygen species level, downregulation of pro-apoptotic activated caspase-3 and Bax, and upregulation of anti-apoptotic Bcl-2, thereby mitigating apoptotic morphological alterations. DPN pre-treatment decreased the expression levels of pro-inflammatory cytokines IL-1β and IL-6 through attenuation of Aβ1–42-induced phosphorylation of mitogen-activated protein kinases JNK and p38. In addition, DPN enhanced ERK1/2 and Akt phosphorylation depressed by Aβ1–42. These findings suggest that DPN protects neurons from Aβ1–42-induced neurotoxicity through a variety of mechanisms, ranging from anti-oxidation, anti-apoptosis, through to anti-inflammation.

Resveratrol inhibits oxygen-glucose deprivation-induced MMP-3 expression and cell apoptosis in primary cortical cells via the NF-κB pathway.

Resveratrol (Res) or trans-3,4',5-trihydroxystilbene, has been proven to exert neuroprotective effects in cerebral ischemia. The aim of the present study was to investigate whether Res has neuroprotective effects in primary cortical neurons subjected to transient oxygen-glucose deprivation(OGD) via inhibiting the expression of the gene encoding stromelysin-1, also known as matrix metalloproteinase-3 (MMP-3), and via inhibiting cell apoptosis. Primary cortical cells were exposed to OGD, followed by reoxygenation to induce transient ischemia. Res (50 µM) was added into the culture medium during transient ischemia in the presence or absence of the nuclear factor(NF)-κB inhibitor pyrrolidine dithiocarbamate(PDTC; 10 µM) or 500 µM of the nitric oxide (NO) donor NOC-18. Cell viability was assessed using the tetrazolium reduction (MTT) assay. Cell apoptosis was evaluated by flow cytometry. MMP-3 expression was analyzed by western blot and reverse transcription-polymerase chain reaction (RT-PCR), while the levels of inducible NO synthase(iNOS), NF-κB, caspase-3, cleaved caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associatedX protein(Bax) were assayed by western blot. NO was detected using a spectrophotometric method. We found that the cellular viability was significantly reduced by transient OGD and that this effect was reversed by Res treatment. In addition, OGD was shown to induce cell apoptosis, the expression of Bax and the activation of caspase-3, and inhibit the expression of Bcl-2, and these effects were also reversed by Res treatment. Res treatment significantly reduced the level of MMP-3 that was induced by transient OGD, via inhibition of NF-κB expression. In addition, Res inhibited iNOS expression and NO synthesis that were induced by OGD. MMP-3 expression induced by NO was attenuated by Res treatment and was partially restored by exogenous NO using NOC-18. Taken together, these findings indicate that OGD induces apoptosis through canonical apoptosis signaling and by modulating the expression of MMP-3; Res can reverse the OGD-induced MMP-3 expression and cell apoptosis via the NF-κB-iNOS/NO pathway. Therefore, Res may be a promising agent for the treatment of neuronal injury associated with stroke.

Orexin A inhibits propofol-induced neurite retraction by a phospholipase D/protein kinase Cε-dependent mechanism in neurons.

BackgroundThe intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction.MethodsIn primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser729 phosphorylation were detected with Western blot.ResultsThe neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser729 phosphorylation, a crucial step in the activation of PKCε.ConclusionsRho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge.

Imprint lithography provides topographical nanocues to guide cell growth in primary cortical cell culture

In this paper, we describe a technology platform to study the effect of nanocues on the cell growth direction in primary cortical cell culture. Topographical cues to cells are provided using nanoscale features created by Jet and Flash Imprint Lithography, coated with polyethylenimine. We investigated nanoscaffolds with periodicities ranging from 200nm to 2000nm, and found that the samples with a period between 400nm and 600nm and a height of 118nm showed highly ordered regions of neurites in a newly formed network with a preferential alignment tendency for astrocytes. Live/dead staining results showed that different materials, such as silicon, glass, and imprinted resist are rendered biocompatible by coating with polyethylenimine. This coating therefore allows for a free choice of scaffold materials and promotes good cell-substrate adhesion. From our results we conclude particular length scales of nanoscaffold can impose a degree of order on cell spreading behavior in a complex cellular brain-on-a-chip network, which could thus be used to emulate ordered brain regions and their function in vitro.

Cerium oxide nanoparticles prevent apoptosis in primary cortical culture by stabilizing mitochondrial membrane potential

Cerium oxide nanoparticles (CNPs) of spherical shape have unique antioxidant capacity primarily due to alternating + 3 and + 4 oxidation states and crystal defects. Several studies revealed the protective efficacies of CNPs in cells and tissues against the oxidative damage. However, its effect on mitochondrial functioning, downstream effectors of radical burst and apoptosis remains unknown. In this study, we investigated whether CNPs treatment could protect the primary cortical cells from loss of mitochondrial membrane potential (Δψm) and Δψm-dependent cell death. CNPs with spherical morphology and size range 7–10 nm were synthesized and utilized at a concentration of 25 nM on primary neuronal culture challenged with 50 μM of hydrogen peroxide (H2O2). We showed that optimal dose of CNPs minimized ROS content of the cells and also curbed related surge in cellular calcium flux. Importantly, CNPs treatment prevented apoptotic loss of cell viability. Reduction in the apoptosis could be successfully attributed to the maintenance of Δψm and restoration of major redox equivalents NADH/NAD+ ratio and cellular ATP. These findings, therefore, suggest possible route of CNPs protective efficacies in primary cortical culture.

Involvement of attenuated antioxidant and Bcl2 signalling property in UV-R/ sunlight irradiated piperine treated ischemia/reperfusion rat model. Highlights

Introduction: Piperine (PIP) is well known multifunctional antioxidant and anti-inflammatory phytochemical that showed neuroprotection. Pre-treatment of UV-R irradiated piperine (UVR-PIP) 10 mg/kg body weight bw, intravenously showed attenuated neuroprotective effects compared to that of PIP (10μM) in PC12, cortical neuronal culture in-vitro and a rat model of focal cerebral ischemia in-vivo. Method: Neurological parameters were evaluated for UVR-PIP and PIP treated against transient focal cerebral ischemia of SD rats through quantification of infarct volume by TTC staining. In-vitro results deal with reduction of cell viability on UVR-PIP treatment in PC12 and cortical neuronal cell. The result of photodegradation of PIP under UV-R irradiation revealed the formation of photoproducts. Estimation of Mitochondrial ROS, antioxidant enzyme and non-enzyme activities in UVR-PIP and PIP treated brain tissue. Results: Results indicated OGD induced primary cortical neuron cultures and PC12 cells showed that SUN-PIP treatment decrease cell viability and increased LDH secretion compared to that of the PIP pre-treatment. In addition, Bcl-2 expression was decreases after the SUN-PIP treatment. Thus, our results demonstrated that SUN-PIP preconditioning failed to increase levels of Bcl-2 and normalized the endogeneous antioxidant, mediating the neuroprotective effects of PIP. UV-R irradiation attenuated the neuroprotective effects of PIP through modulation of the antioxidant and Bcl-2 mediated pathway. Conclusion: Thus, the ability of UV-R to serve as a modulator of this neuroprotective signaling pathway. Piperine loses some of its neuroprotective ability when irradiated by UV radiation and care has to be taken during storage to avoid exposing piperine to the sun.

Piracetam ameliorated oxygen and glucose deprivation-induced injury in rat cortical neurons via inhibition of oxidative stress, excitatory amino acids release and P53/Bax.

Our previous work has demonstrated that piracetam inhibited the decrease in amino acid content induced by chronic hypoperfusion, ameliorated the dysfunction of learning and memory in a hypoperfusion rat model, down-regulated P53, and BAX protein, facilitated the synaptic plasticity, and may be helpful in the treatment of vascular dementia. To explore the precise mechanism, the present study further evaluated effects of piracetam on Oxygen and glucose deprivation (OGD)-induced neuronal damage in rat primary cortical cells. The addition of piracetam to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release experiments. Piracetam also lowered the levels of malondialdehyde, nitrogen monoxidum, and xanthine oxidase which was increased in the OGD cells, and enhanced the activities of superoxide dismutase and glutathione peroxidase, which were decreased in the OGD cells. We also demonstrated that piracetam could decrease glutamate and aspartate release when cortical cells were subjected to OGD. Furthermore, Western blot study demonstrated that piracetam attenuated the increased expression of P53 and BAX protein in OGD cells. These observations demonstrated that piracetam reduced OGD-induced neuronal damage by inhibiting the oxidative stress and decreasing excitatory amino acids release and lowering P53/Bax protein expression in OGD cells.

Nucleoside 5′-phosphorothioate derivatives are highly effective neuroprotectants

The brain is especially sensitive to oxidative stress due to its high rate of oxidative metabolism, relatively low levels of antioxidant enzymes, and high concentrations of Fe/Cu ions. During the neurodegeneration process, the aggregation of proteins Aβ, accompanies oxidative stress. We explored the potential of thiophosphate derivatives to rescue neurons from oxidative stress and Aβ toxicity. We evaluated the neuroprotective effect of ATP-γ-S, ADP-β-S, and GDP-β-S on primary cortical neuronal cells exposed to several insults, including treatment with FeSO4, co-application of H2O2 and FeSO4, and addition of Aβ42. Upon treatment with FeSO4, phosphorothioate analogues exhibited up to 3000-fold better neuroprotectant activity than the corresponding parent nucleotides. Likewise, phosphorothioate analogues proved to be up to 30-fold better neuroprotectants than the corresponding parent nucleotides upon treatment with both H2O2 and FeSO4. When we exposed primary neuron and astrocyte cultures to 50μM Aβ42-induced cell death, we found that ATP-γ-S significantly improved cell morphology and maintained culture viability with an IC50 value of 0.8μM. Finally, we evaluated the viability of neuroblastoma cells under hypoxic conditions in the presence of ATP-γ-S and found that the latter was involved in the regulation of HIF-1a and stabilized mRNA levels of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1), which promote cell survival and proliferation. Based on its high potency as a neuroprotectant, we propose ATP-γ-S as a highly promising, biocompatible, and water-soluble drug candidate for the treatment of neurodegenerative disorders.

Abstract W P216: Inducible Glutamate Oxaloacetate Transaminase: A New Target for Stroke Therapy

Introduction: Elevated extracellular glutamate plays a central role in mediating ischemic brain damage. The current work stems from our key observation demonstrating that induction of glutamate oxaloacetate transaminase (GOT), a glutamate metabolizing enzyme in the brain, can attenuate stroke-induced injury. At the stroke site, inducible GOT (iGOT) catalyzes the metabolism of extracellular glutamate via a truncated TCA cycle to generate energy and limit concentrations. Hypotheses: Phytoestrogen Biochanin induces GOT. iGOT inducer Biochanin protects against stroke in vivo. Methods: In vitro. Primary cortical neuronal cells were isolated from the cerebral cortex of C57/BL6 mice feti and seeded in 12-well plates maintained for 24h under standard media and culture conditions prior to experimental treatment. After 24h, cells were incubated with vehicle control (DMSO) or one of several screened phytoestrogen isoflavones for 72h. After 72h sample were collected for mRNA and protein expression analyses of GOT. In vivo. Mice (C57/BL6) received daily IP injection of vehicle control (75% DMSO in water, n=4) or Biochanin A (10mg/kg body weight, n=6) for 3 weeks prior to 90min middle cerebral artery occlusion (MCAO). Following 48h, mice were subjected to 11.7T MRI for determination of stroke induced lesion volume and brain tissue were collected for downstream mRNA and protein expression analyses of GOT. Results: The following phytoestrogen isoflavones were screened for their ability to induce GOT: biochanin A, flavaprenin, diadzein, formononetin, glycitin, and genistein. Biochanin A was identified as a potent (3-fold) inducer of GOT. Biochanin A significantly induced GOT mRNA expression in neural cells. In an in vivo setting of acute ischemic stroke, Biochanin A treated mice had significantly (n=6; p&lt;0.05) smaller stroke-induced lesions as compared to vehicle controls. Conclusion: In this work we have identified a natural small molecule inducer of GOT - Biochanin A. Biochanin A is a safe, naturally occurring phytoestrogen isoflavone that represents a therapeutic target that lends itself to be translated to clinical study.

Hydroxy-safflor yellow A inhibits neuroinflammation mediated by Aβ1–42 in BV-2 cells

Inflammation is an important contributor to the development of Alzheimer's disease (AD). Anti-inflammatory medication may offer promising treatment for AD. Hydroxy-safflor yellow A (HSYA), a chemical component of the safflower yellow pigments, has been reported to exert potent immunosuppressive effects. This study examined the anti-inflammatory effects of HSYA in Aβ1–42-treated BV-2 microglia cells. The mRNA levels of IL-1β, IL-4, IL-10, TNF-α, COX-2 and iNOS were detected by real-time PCR. Western blotting was used to determine the protein expression of COX-2, TNF-α, iNOS, Janus Kinase 2 (JAK2), p-JAK2, signal transducers and activators of transcription 3 (STAT3) and p-STAT3. BV2-conditioned medium was used to treat SH-SY5Y cells and primary neuronal cells in indirect toxicity experiments. Cell viability and apoptosis were assessed using MTT assay and Annexin V/PI staining respectively. The results demonstrated that HSYA significantly reduced the expression of the pro-inflammatory mediators and inhibited Aβ1–42-induced neuroinflammation. Moreover, HSYA protected primary cortical neurons and SH-SY5Y cells against microglia-mediated neurotoxicity. HSYA also enhanced the phosphorylation of JAK2/STAT3 pathway and inhibition of JAK2 by AG 490 attenuated the anti-inflammatory effects of HSYA. Overall, our findings suggested that HSYA inhibited Aβ1–42-induced inflammation and conferred neuroprotection partially through JAK2/STAT3 pathway, indicating that HSYA could be a potential drug for the treatment of AD.

NMDA receptor-mediated neuroprotective effect of the Scutellaria baicalensis Georgi extract on the excitotoxic neuronal cell death in primary rat cortical cell cultures.

The objective of the current research work was to evaluate the neuroprotective effect of the ethanol extract of Scutellaria baicalensis (S.B.) on the excitotoxic neuronal cell death in primary rat cortical cell cultures. The inhibitory effects of the extract were qualitatively and quantitatively estimated by phase-contrast microscopy and lactate dehydrogenase (LDH) assays. The extract exhibited a potent and dose-dependent inhibition of the glutamate-induced excitotoxicity in the culture media. Further, using radioligand binding assays, it was observed that the inhibitory effect of the extract was more potent and selective for the N-methyl-D-aspartate (NMDA) receptor-mediated toxicity. The S.B. ethanol extract competed with [3H] MDL 105,519 for the specific binding to the NMDA receptor glycine site with 50% inhibition occurring at 35.1 μg/mL. Further, NMDA receptor inactivation by the S.B. ethanol extract was concluded from the decreasing binding capability of [3H]MK-801 in the presence of the extract. Thus, S.B. extract exhibited neuroprotection against excitotoxic cell death, and this neuroprotection was mediated through the inhibition of NMDA receptor function by interacting with the glycine binding site of the NMDA receptor. Phytochemical analysis of the bioactive extract revealed the presence of six phytochemical constituents including baicalein, baicalin, wogonin, wogonoside, scutellarin, and Oroxylin A.

Film-based Implants for Supporting Neuron-Electrode Integrated Interfaces for The Brain.

Neural engineering provides promise for cell therapy by integrating the host brain with brain-machine-interface technologies in order to externally modulate functions. Long-term interfaces with the host brain remain a critical challenge due to insufficient graft cell survivability and loss of brain electrode sensitivity over time. Here, integrated neuron-electrode interfaces were developed on thin flexible and transparent silk films as brain implants. Mechanical properties and surface topography of silk films were optimized to promote cell survival and alignment of primary rat cortical cells. Compartmentalized cultures of living neural circuit and co-patterned electrode arrays were incorporated on the silk films with built-in wire connections. Electrical stimulation via electrodes embedded in the films activated surrounding neurons evoked calcium responses. In mice brains the silk film implants showed conformal contact capable of modulating host brain cells with minimal inflammatory response and stable indwelling for weeks. The approach of combining cell therapy and brain electrodes could provide sustained functional brain-machine interfaces with ex vivo control of neuron-electrode interface with spatial and temporal precision.

Endothelial Nitric Oxide Synthase Regulates White Matter Changes via the BDNF/TrkB Pathway after Stroke in Mice

Stroke induced white matter (WM) damage is associated with neurological functional deficits, but the underlying mechanisms are not well understood. In this study, we investigate whether endothelial nitric oxide synthase (eNOS) affects WM-damage post-stroke. Adult male wild-type (WT) and eNOS knockout (eNOS-/-) mice were subjected to middle cerebral artery occlusion. Functional evaluation, infarct volume measurement, immunostaining and primary cortical cell culture were performed. To obtain insight into the mechanisms underlying the effects of eNOS-/- on WM-damage, measurement of eNOS, brain-derived neurotrophic factor (BDNF) and its receptor TrkB in vivo and in vitro were also performed. No significant differences were detected in the infarction volume, myelin density in the ipsilateral striatal WM-bundles and myelin-based protein expression in the cerebral ischemic border between WT and eNOS-/- mice. However, eNOS-/- mice showed significantly: 1) decreased functional outcome, concurrent with decreases of total axon density and phosphorylated high-molecular weight neurofilament density in the ipsilateral striatal WM-bundles. Correlation analysis showed that axon density is significantly positive correlated with neurological functional outcome; 2) decreased numbers of oligodendrocytes / oligodendrocyte progenitor cells in the ipsilateral striatum; 3) decreased synaptophysin, BDNF and TrkB expression in the ischemic border compared with WT mice after stroke (n = 12/group, p<0.05). Primary cortical cell culture confirmed that the decrease of neuronal neurite outgrowth in the neurons derived from eNOS-/- mice is mediated by the reduction of BDNF/TrkB (n = 6/group, p<0.05). Our data show that eNOS plays a critical role in WM-damage after stroke, and eNOS-/--induced decreases in the BDNF/TrkB pathway may contribute to increased WM-damage, and thereby decrease functional outcome.

N-benzylcinnamide protects rat cultured cortical neurons from β-amyloid peptide-induced neurotoxicity

The pathogenesis of Alzheimer's disease involves an amyloid β-peptide (Aβ)-induced cascade of elevated oxidative damage and inflammation. The present study investigates the protective effects and the underlying mechanisms of N-benzylcinnamide (PT-3), purified from Piper submultinerve. Against Aβ-induced oxidative stress and inflammation in rat primary cortical cell cultures. Pre-treatment with 10–00nM PT-3 significantly attenuated neuronal cell death induced by 10μM Aβ1–42. PT-3 was found to enhance cell viability through a significant reduction in the level of reactive oxygen species, down-regulated expression of pro-apoptotic activated caspase-3 and Bax, increased expression of anti-apoptotic Bcl-2, and mitigation of Aβ-induced morphological alterations. Regarding its effects on inflammatory responses, PT-3 pre-treatment decreased the expression of pro-inflammatory cytokines IL-1β and IL-6. The mechanisms of PT-3 neuronal protection against inflammation may be associated with the mitogen-activated protein kinases (MAPK) pathway. Aβ1–42-induced phosphorylation of JNK and p38 MAPK was inhibited by pretreatment with PT-3 in a dose-dependent manner. However, phosphorylation of ERK1/2 was not affected by either PT-3 or Aβ1–42. PT-3 did not stimulate Akt phosphorylation, which was inhibited by Aβ1–42. These findings suggest that PT-3 protects neurons from Aβ1–42-induced neurotoxicity through its anti-apoptotic, anti-oxidative, and anti-inflammatory properties with inhibition of JNK and p38 MAPK phosphorylation as the potential underlying mechanism.

Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells

Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased mitochondrial delivery to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1-light chain-3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modeling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an “eat-me” signal for the elimination of damaged mitochondria from neuronal cells.

Mesenchymal Stem Cells from Human Umbilical Cord Express Preferentially Secreted Factors Related to Neuroprotection, Neurogenesis, and Angiogenesis

Mesenchymal stem cells (MSCs) are promising tools for the treatment of diseases such as infarcted myocardia and strokes because of their ability to promote endogenous angiogenesis and neurogenesis via a variety of secreted factors. MSCs found in the Wharton’s jelly of the human umbilical cord are easily obtained and are capable of transplantation without rejection. We isolated MSCs from Wharton’s jelly and bone marrow (WJ-MSCs and BM-MSCs, respectively) and compared their secretomes. It was found that WJ-MSCs expressed more genes, especially secreted factors, involved in angiogenesis and neurogenesis. Functional validation showed that WJ-MSCs induced better neural differentiation and neural cell migration via a paracrine mechanism. Moreover, WJ-MSCs afforded better neuroprotection efficacy because they preferentially enhanced neuronal growth and reduced cell apoptotic death of primary cortical cells in an oxygen-glucose deprivation (OGD) culture model that mimics the acute ischemic stroke situation in humans. In terms of angiogenesis, WJ-MSCs induced better microvasculature formation and cell migration on co-cultured endothelial cells. Our results suggest that WJ-MSC, because of a unique secretome, is a better MSC source to promote in vivo neurorestoration and endothelium repair. This study provides a basis for the development of cell-based therapy and carrying out of follow-up mechanistic studies related to MSC biology.