Abstract WMP35: Low Level Laser Therapy (LLLT) Protects Against Oxygen-Glucose Deprivation-Induced Neuron Death by Modulating Nitric Oxide and ROS Production In Vitro

Stroke ◽  
2013 ◽  
Vol 44 (suppl_1) ◽  
Author(s):  
Zhanyang Yu ◽  
Ning Liu ◽  
Yadan Li ◽  
Jianhua Zhao ◽  
Eng H Lo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Cell Survival ◽  
Cortical Neurons ◽  
Post Treatment ◽  
Low Level Laser Therapy ◽  
Primary Neurons ◽  
Ros Production ◽  
Primary Mouse ◽  
Survival Signaling

Background: LLLT has been shown to be neurorprotective against cerebral ischemia in animals. Initial human clinical trials demonstrated safety and potential beneficial profiles. A pivotal clinical trial with TLT is ongoing for ischemic stroke. However, the underlying neuroprotective mechanisms require further definition. We previously showed LLLT protected primary neurons against OGD. In the current study, we tested the hypothesis that LLLT may attenuate OGD-induced nitric oxide (NO) and ROS production and associated cell survival signaling, causing reduced cell death in primary mouse cortical neurons. Method: At day 8 of culture, neurons were subjected to 4 hr OGD. LLLT was applied at 2 hr reoxygenation. NO level, nNOS expression, ROS level, and cell survival signaling were measured post-LLLT. Cell death was measured at 20 hr after OGD. Results were expressed as fold of normal control, mean ± SEM, n = 3-5 per group, P <0.05 was considered statistically significant. Results: OGD significantly increased NO level in primary neurons (2.12 ± 0.47, 2.67 ± 1.07, and 2 ± 0.77 at 5 min, 30 min and 1 hr post-treatment, respectively), but the increase was significantly reduced by LLLT (1.08 ± 0.44 at 5 min and 1.45 ± 0.73 at 30 min post-LLLT). OGD also significantly increased nNOS mRNA level (2.17 ± 0.42, 3.99 ± 1.7, and 2.60 ± 0.7 at 5 min, 30 min and 1 hr post-treatment, respectively), while the increase was significantly ameliorated by LLLT (0.50 ± 0.08 at 30 min and 0.34 ± 0.45 at 1 hr post-LLLT). Additionally, 50 uM NO donor SNAP exposure for 48 hr induced 23.6 ± 2.5% cell death, which was significantly ameliorated by LLLT (12.3 ± 4.2% cell death), indicating LLLT may directly protect NO-mediated neurotoxicity. Furthermore, LLLT significantly reduced OGD-induced ROS production at 5 min, 30 min and 1 hr post-LLLT. Finally, OGD suppressed neuron survival signaling p-Akt and Bcl-2 protein expressions, but they were significantly rescued by LLLT. Conclusion: These results suggest LLLT protection against OGD may be partially through reducing OGD-induced NO and ROS production, and restoring OGD-suppressed cell survival signaling.

scholarly journals A microscopy-based small molecule screen in primary neurons reveals neuroprotective properties of the FDA-approved anti-viral drug Elvitegravir

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Simon F. Merz ◽  
C. Peter Bengtson ◽  
Clara Tepohl ◽  
Anna M. Hagenston ◽  
Hilmar Bading ◽  
...  
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Nmda Receptor ◽  
Mitochondrial Membrane Potential ◽  
Cortical Neurons ◽  
Mitochondrial Membrane ◽  
Glutamate Toxicity ◽  
Primary Neurons ◽  
Screening Assay ◽  
Primary Mouse

Abstract Glutamate toxicity is a pathomechanism that contributes to neuronal cell death in a wide range of acute and chronic neurodegenerative and neuroinflammatory diseases. Activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor and breakdown of the mitochondrial membrane potential are key events during glutamate toxicity. Due to its manifold functions in nervous system physiology, however, the NMDA receptor is not well suited as a drug target. To identify novel compounds that act downstream of toxic NMDA receptor signaling and can protect mitochondria from glutamate toxicity, we developed a cell viability screening assay in primary mouse cortical neurons. In a proof-of-principle screen we tested 146 natural products and 424 FDA-approved drugs for their ability to protect neurons against NMDA-induced cell death. We confirmed several known neuroprotective drugs that include Dutasteride, Enalapril, Finasteride, Haloperidol, and Oxybutynin, and we identified neuroprotective properties of Elvitegravir. Using live imaging of tetramethylrhodamine ethyl ester-labelled primary cortical neurons, we found that Elvitegravir, Dutasteride, and Oxybutynin attenuated the NMDA-induced breakdown of the mitochondrial membrane potential. Patch clamp electrophysiological recordings in NMDA receptor-expressing HEK293 cell lines and primary mouse hippocampal neurons revealed that Elvitegravir does not act at the NMDA receptor and does not affect the function of glutamatergic synapses. In summary, we have developed a cost-effective and easy-to-implement screening assay in primary neurons and identified Elvitegravir as a neuro- and mitoprotective drug that acts downstream of the NMDA receptor.

Abstract 3115: Low-Level Laser Therapy Protects Against OGD-induced Neurotoxicity Of Primary Cultured Mouse Cortical Neurons

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Zhanyang Yu ◽  
Ning Liu ◽  
Eng H Lo ◽  
Thomas J McCarthy ◽  
Xiaoying Wang
Keyword(s):  
Laser Therapy ◽  
Mitochondrial Function ◽  
Cortical Neurons ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Low Level Laser Therapy ◽  
Primary Neurons ◽  
Novel Therapy ◽  
Low Level ◽  
Mtt Reduction

Background: Low level light (or laser) therapy (LLLT) has been studied and practiced for promoting wound healing, reducing pain, inflammation, and ischemic tissue damage. Recently, a series of experimental and clinical investigations have suggested that LLLT may be a novel therapy against hypoxic/ischemic brain damage. A clinical trial of LLLT therapy for ischemic stroke is now on going. However, the molecular mechanism of LLLT-conferred neuroprotection remains poorly defined. In this study, we tested our hypothesis that LLLT may attenuate impairments of mitochondrial function induced by hypoxic/ischemic insults in primary cultured mouse cortical neurons. Method: At day 9 of culture, primary neurons were subjected to 4 hr OGD followed by reoxygenation. One 810-nm LLLT treatment was applied for 2 minutes at 2 hr after reoxygenation. Neurotoxicity was measured after 20 hr after reoxygenation by LDH release assay. We also measured MTT reduction and mitochondria membrane potential (MMP) at 2 hr after LLLT treatment as markers of mitochondrial function. Results: The neurotoxicity study showed that 4 hr OGD plus 20 hr reoxygenation caused 33.8± 3.4% neuronal cell death, while LLLT treatment significantly reduced the neuronal death rate to 23.6± 2.9% (30.2% reduction, n=6, p smaller than 0.05). Mitochondrial functional assays showed OGD decreased MTT reduction to 75.9± 2.68%, but LLLT treatment significantly rescued MTT reduction to 87.6±4.55% (15.4% improvement, n=6, p smaller than 0.05). Furthermore, after OGD, MMP was reduced to 48.9±4.39%, while LLLT treatment significantly ameliorated this reduction to 89.6± 13.9% (83% improvement, n=4, p smaller than 0.05) compared to normoxic controls. Conclusion: The present study suggests that LLLT treatment is protective against OGD-induced neurotoxicity of primary neurons and that this protection may be conferred through preservation or rescue of mitochondrial function.

scholarly journals Nitric Oxide Inhibits Caspase Activation and Apoptotic Morphology but Does Not Rescue Neuronal Death

2005 ◽  
Vol 25 (3) ◽  
pp. 348-357 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Costantino Iadecola
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Survival ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Cysteine Residue ◽  
Caspase Activity ◽  
No Donors ◽  
Apoptotic Morphology

Nitric oxide (NO) has been shown to inhibit apoptotic cell death by S-nitrosylation of the catalytic-site cysteine residue of caspases. However, it is not clear whether in neurons NO-mediated caspase inactivation leads to improved cell survival. To address this issue, we studied the effect of NO donors on caspase activity and cell survival in cortical neuronal culture treated with the apoptosis inducer staurosporine (STS) and camptothecin. In parallel, cell viability was assessed by the MTS assay and MAP2 staining. We found that NO donors ((±)- S-nitroso- N-acetylpenicillamine, S-nitrosoglutathione, and NONOates) dose-dependently inhibited caspase-3 and -9 activity induced by STS and camptothecin. The reduction in caspase-3 activity was, in large part, because of the blockage of the proteolytic conversion of pro-caspase-3 to active caspase-3. NO donors also inhibited the appearance of the classical apoptotic nuclear morphology. However, inhibition of both caspase activity and apoptotic morphology was not associated with enhancement of cell viability. Thus, inhibition of caspase and apoptotic morphology by NO donors does not improve neuronal survival. The data suggest that inhibition of caspase by NO unmasks a caspase-independent form of cell death. A better understanding of this form of cell death may provide new strategies for neuroprotection in neuropathologies, such as ischemic brain injury, associated with apoptosis.

scholarly journals Effect and Mechanism of 808 nm Light Pretreatment of Hypoxic Primary Neurons

2014 ◽  
Vol 2014 ◽  
pp. 1-5
Author(s):  
Xin Chen ◽  
Qiang-Li Wang ◽  
Shuang Li ◽  
Xian-qiang Mi
Keyword(s):  
Cell Viability ◽  
Cortical Neurons ◽  
Colorimetric Assay ◽  
Neuronal Morphology ◽  
Light Irradiation ◽  
Control Group ◽  
Primary Neurons ◽  
Atp Content ◽  
Low Intensity ◽  
Primary Mouse

This study investigated the effect of low intensity 808 nm light pretreatment of hypoxic primary neurons. Cobalt chloride (CoCl2) has been used to induce hypoxic injury in primary mouse cortical neurons. Low intensity 808 nm light was from light-emitting diode (LED). Cells were randomly divided into 4 groups: normal control group, CoCl2-induced group, CoCl2-induced group with 808 nm light irradiation pretreatment, and normal group with 808 nm light irradiation pretreatment. Effect of low intensity 808 nm light on neuronal morphology has been observed by microscope. MTT colorimetric assay has been used to detect the effect of low intensity 808 nm light on neuronal activity. Adenosine triphosphate (ATP) concentration and cytochrome C oxidase (COX) activity has been detected to study the effect of low intensity 808 nm light on neuronal mitochondria function. The results indicated that low intensity 808 nm light pretreatment alone did not affect cell viability, COX activity, and ATP content of neurons and low intensity 808 nm light pretreatment promoted the cell viability, COX activity, and ATP content of neurons with CoCl2exposure; however, low intensity 808 nm light pretreatment did not completely recover COX activity and cellular ATP content of primary neurons with CoCl2exposure to the level of the normal neurons.

scholarly journals Paraquat Modulates Immunological Function in Bone Marrow-Derived Macrophages

2021 ◽  
Author(s):  
Piyarat Srinont ◽  
Jaroon Wandee ◽  
Worapol Angwanich
Keyword(s):  
Nitric Oxide ◽  
Reactive Oxygen Species ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Viability ◽  
Inducible Nitric Oxide Synthase ◽  
Ros Production ◽  
Oxygen Species ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Abstract Paraquat (PQ) is an herbicide commonly used worldwide. This herbicide is known to alter the human and animal immune systems. Many reports indicated that PQ impacts immune cell viability and functions. However, the underlying mechanism critical is still unknown. Therefore, the aim of this study was to evaluate effects of PQ on free radical production, oxidative stress, cell death, and pro-inflammatory gene expression of murine bone marrow-derived macrophages (BMDMs) from female C57BL/6NJcl mice in vitro. BMDMs were incubated with PQ at 0, 200, 400 µM for 24 h. Intracellular reactive oxygen species (ROS) production, apoptosis, cell viability, nitric oxide, inducible nitric oxide synthase (iNOS), and IL-6 expression of murine BMDMs were measured. The results revealed that PQ treatments led to decrease the cell viability and induced apoptotic cell death in a dose-dependent manner. Additionally, PQ induced reactive oxygen species (ROS) generation. The mRNA expression level of pro-inflammatory mediator gene IL-6 and inducible nitric oxide synthase (iNOS) were elevated, while the level of lipid peroxides (MDA) production was unaltered by PQ treatment. Interestingly, PQ led to a decrease in nitric oxide production depends on its concentration. These phenomena indicated that PQ increased cellular ROS production which induced apoptosis, and the herbicide triggers production of iNOS and IL-6 in murine BMDMs.

scholarly journals The Gtpase Rho Controls a P53-Dependent Survival Checkpoint during Thymopoiesis

2000 ◽  
Vol 192 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Patrick S. Costello ◽  
Steve C. Cleverley ◽  
Ricciarda Galandrini ◽  
Stefan W. Henning ◽  
Doreen A. Cantrell
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Survival ◽  
Ectopic Expression ◽  
Double Positive ◽  
Cell Stage ◽  
Guanine Nucleotide Binding Protein ◽  
Survival Signaling ◽  
T Cell Progenitors

During the early stages of thymopoiesis, cell survival is controlled by cytokines that regulate the expression of antiapoptotic proteins such as Bcl-2. At the pre-T cell stage, a critical checkpoint for β chain selection is monitored by the tumor suppressor p53: pre-T cells can survive and differentiate when p53 is removed genetically or when its proapoptotic function is inactivated physiologically as a consequence of signaling through the pre-T cell receptor complex. Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors. Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of β selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53. The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity. Further analysis revealed that loss of Rho function caused survival defects in CD4/8 double-positive thymocytes that is independent of p53 but can be prevented by ectopic expression of Bcl-2. These studies highlight that the GTPase Rho is a crucial component of survival signaling pathways in at least two different thymocyte subpopulations: Rho controls the p53 survival checkpoint in pre-T cells and is also crucial for a p53 independent survival signaling pathway in CD4/8 double positives.

scholarly journals p38 Map Kinase Mediates Bax Translocation in Nitric Oxide–Induced Apoptosis in Neurons

2000 ◽  
Vol 150 (2) ◽  
pp. 335-348 ◽  
Author(s):  
Saadi Ghatan ◽  
Stephen Larner ◽  
Yoshito Kinoshita ◽  
Michal Hetman ◽  
Leena Patel ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Map Kinase ◽  
Cortical Neurons ◽  
Kinase Activity ◽  
Critical Role ◽  
Primary Cultures ◽  
P38 Map Kinase ◽  
Concomitant Treatment ◽  
Cell Death Pathway

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, we determined if NO-induced cell death in neurons was dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), an NO donor, elevated caspase activity and induced death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminished caspase induction and protected SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk did not provide significant protection. A role for p38 MAP kinase was further substantiated by the observation that SB203580 blocked translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promoted Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons were resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway.

scholarly journals Survival Signaling by C-RAF: Mitochondrial Reactive Oxygen Species and Ca2+ Are Critical Targets

2008 ◽  
Vol 28 (7) ◽  
pp. 2304-2313 ◽  
Author(s):  
Andrey V. Kuznetsov ◽  
Julija Smigelskaite ◽  
Christine Doblander ◽  
Manickam Janakiraman ◽  
Martin Hermann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Cell Death ◽  
Reactive Oxygen ◽  
Ros Production ◽  
Oxygen Species ◽  
Mitochondrial Reactive Oxygen Species ◽  
Mitochondrial Ros ◽  
Survival Signaling ◽  
First Time

ABSTRACT Survival signaling by RAF occurs through largely unknown mechanisms. Here we provide evidence for the first time that RAF controls cell survival by maintaining permissive levels of mitochondrial reactive oxygen species (ROS) and Ca2+. Interleukin-3 (IL-3) withdrawal from 32D cells resulted in ROS production, which was suppressed by activated C-RAF. Oncogenic C-RAF decreased the percentage of apoptotic cells following treatment with staurosporine or the oxidative stress-inducing agent tert-butyl hydroperoxide. However, it was also the case that in parental 32D cells growing in the presence of IL-3, inhibition of RAF signaling resulted in elevated mitochondrial ROS and Ca2+ levels. Cell death is preceded by a ROS-dependent increase in mitochondrial Ca2+, which was absent from cells expressing transforming C-RAF. Prevention of mitochondrial Ca2+ overload after IL-3 deprivation increased cell viability. MEK was essential for the mitochondrial effects of RAF. In summary, our data show that survival control by C-RAF involves controlling ROS production, which otherwise perturbs mitochondrial Ca2+ homeostasis.

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