Oxidative Stress Induces Apoptosis in Rat Cerebral Cortical Cultures：Different Responses in Neurons and Glia
| Autor: | JIZ-YUH WANG, 王志煜 |
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| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 Oxidative stress in the brain has been increasingly associated with the development of numerous human neurodegenerative diseases in which apoptosis is implicated. Microglia, which play a pivotal role in immunological defense, readily become activated upon neuronal injury or inflammatory stimulation and release immune modulators such as cytokines, chemokines as well as the potentially damaging nitric oxide (NO) and reactive oxygen species (ROS), including superoxide anion (•O2), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), thereby further contributing to oxidative neurotoxicity. The reaction between NO and •O2 forming toxic peroxynitrite anion (ONOO) has been proposed to play a major pathogenic role in neuronal injury. However, little is known concerning the fate of activated glia. Moreover, the interactions between H2O2 and NO during oxidative stress in the brain, which may promote or diminish cell death, is also less clear. In this study, we proposed to induce oxidative neurotoxicity in vitro indirectly by endotoxin lipopolysaccharide (LPS) or high glucose and directly by H2O2 or NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP), and explored the responses of rat cerebral cortex neurons and glia in primary cultures. Additionally, the capacities of free radical scavengers, including superoxide dimutase (SOD)、catalase、2, 2, 6, 6-tetramethyl-1-piperidinoxyl (TEMPO)、N-acetylcysteine (NAC)、dimethylthiourea (DMTU)、uric acid (UA) and 17-estradiol, on sweeping out NO、ROS and ONOO, and against oxidative neurotoxicity were also examined. In the experiments of primary glial cultures stimulated by LPS or high glucose, mixed glia exposed to increased concentrations of LPS showed excellent concentration-response correlation on productions of NO and tumor necrosis factor alpha (TNF-). No injury was seen in mixed glia following the maximal LPS stimulation (=10000 ng/ml). Microglia treated with 1 ng/ml LPS resulted in the maximal response and activation as measured by the release of NO and TNF-. However, treatment with higher concentrations of LPS significantly resulted in lower quantities of detectable TNF-. Further analysis revealed that overactivation of microglia treated with LPS at concentrations over 100 ng/ml resulted in a time- and concentration-dependent apoptotic death as defined by DNA strand breaks and surface expression of apoptosis-specific marker phosphatidylserine (PS). Moreover, glucose concentration-dependently (25-125 mM) generated cytotoxicity, while synergistic apoptosis of microglia was found following the treatment of LPS in the presence of increased levels of glucose. This synergistic cytotoxicity was attenuated by the use of SOD and catalase, suggesting the involvement of ROS. Contrast to microglia, astrocytes did not produce NO and were completely insensitive to cytotoxicity following LPS stimulation. In the experiments of primary glial or neuronal cultures facing oxidative stresss induced by H2O2 or SNAP, results indicated that H2O2 and SNAP by themselves elicited cell death, such as neurons, mixed glia, microglia and astrocytes, in a concentration-dependent manner. Pre-incubation with catalase、TEMPO、DMTU、17-estradiol or 17-estradiol, but not SOD, significantly reduced oxidative neurotoxicity induced by H2O2 in mixed glia. Furthermore, the inhibitory effect of 17-estradiol on H2O2-induced oxytoxicity could not be interfered by tamoxifen (estrogen receptor antagonist), suggesting that 17-estradiol promoted cell survival under oxidative stress through its own antioxidant property. Additionally, SNAP at concentrations of 30-100 M may rapidly attenuate H2O2-induced oxytoxicity in mixed glia and microglia, but not in astrocytes. Sublytic concentrations (=100 M) of H2O2 plus SNAP were sufficient to induce neuronal apoptosis as determined by DNA laddering and fluorescent staining of apoptotic nuclei. As the mechanisms may involve reactions between H2O2 and NO, we monitored the production of ONOO and ROS throughout the experiments and found that transient ONOO increase was accompanied by rapid H2O2 decay and NO production, whereas ROS slowly decreased following treatment. Furthermore, p38 mitogen-activated protein kinase (p38 MAPK) activation and the cleavage of caspase-3 were observed. Conversely, inhibition of p38 MAPK and caspase-3 significantly reduced apoptotic death induced by H2O2+SNAP. Free radical scavengers, such as catalase、TEMPO、DMTU and NAC, excepting SOD and UA, protected neurons against H2O2+SNAP-induced oxidative neurotoxicity. Both TEMPO and NAC exhibited significant capacities in scavenging NO、ROS and ONOO. Collectively, the experimental findings represented here suggest that overactivation-induced apoptosis of microglia may serve as a negatively feedback modulation on inflammatory fierceness in brain and avoid bystander killing of neurons. Increased ambient levels of glucose (e.g. hyperglycemia) renders microglia vulnerable to inflammatory insults, and leads to a synergistic apoptosis. NO, acting as an antioxidant, attenuates H2O2-induced oxytoxicity occurred in glia. In contrary, H2O2 and NO act synergistically to induce neuronal death through apoptosis in which activation of p38 MAPK and caspase-3 is involved. In this study, elucidation of the neuroprotective properties of free radical scavengers and the pathological mechanisms of oxidative neurotoxicity may provide new insight into a novel therapeutic intervention on chronic neurological disorders. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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