Microglial Phagocytosis of Dopamine Neurons at Early Phases of Apoptosis

Transection of the medial forebrain bundle caused apoptosis of dopamine neurons in the rat substantia nigra. Immunohistochemical localization of activated microglia and tyrosine hydroxylase in the axotomized substantia nigra showed that activation of microglia was rapid and OX-6 (MHC-II marker)-positive and ED1 (lysosomal phagocytic marker)-positive microglia were apposed to structurally intact tyrosine hydroxylase-positive dopamine neurons, indicating microglial phagocytosis of degenerating dopamine neurons. The occurrence of microglial phagocytosis at early stages of apoptosis may indicate the evolution of apoptosis into an irreversible state. Alternatively, interventions that suppress early activation of microglia might lead to novel mechanisms for neuron protection.     

Microglia activation-induced mesencephalic dopaminergic neurodegeneration — an in vitro model for Parkinson’s disease

Uncontrolled and chronic microglia activation has been implicated in the process of dopaminergic neuron degeneration in sporadic Parkinson??s disease (PD). Elevated proinflammatory mediators, presumably from activated microglia (e.g., cytokines, PGE₂, nitric oxide, and superoxide radical), have been observed in PD patients and are accompanied by dopaminergic neuronal loss. Preclinical studies have demonstrated the deleterious effects of proinflammatory mediators in various in vivo and in vitro models of PD. The use of in vitro studies provides a unique tool to investigate the interaction between neurons and microglia and is especially valuable when considering the role of activated microglia in neuronal death. Here we summarize findings highlighting the potential mechanisms of microgliamediated neurodegeneration in PD.     

Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo

MPTP produces clinical, biochemical, and neuropathologic changes reminiscent of those that occur in idiopathic Parkinson's disease (PD). In the present study we show that MPTP treatment led to activation of microglia in the substantia nigra pars compacta (SNpc), which was associated and colocalized with an increase in inducible nitric oxide synthase (iNOS) expression. In iNOS-deficient mice the increase of iNOS expression but not the activation of microglia was blocked. Dopaminergic SNpc neurons of iNOS-deficient mice were almost completely protected from MPTP toxicity in a chronic paradigm of MPTP toxicity. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites did not differ between iNOS-deficient mice and their wild-type littermates, this protection was not associated with a preservation of nigrostriatal terminals. Our results suggest that iNOS-derived nitric oxide produced in microglia plays an important role in the death of dopaminergic neurons but that other mechanisms contribute to the loss of dopaminergic terminals in MPTP neurotoxicity. We conclude that inhibition of iNOS may be a promising target for the treatment of PD.     

Liposome-delivered superoxide dismutase prevents nitric oxide-dependent motor neuron death induced by trophic factor withdrawal

Inhibition of nitric oxide synthesis prevents rat embryonic motor neurons from undergoing apoptosis when initially cultured without brain-derived neurotrophic factor. Using an improved cell culture medium, we found that the partial withdrawal of trophic support even weeks after motor neurons had differentiated into a mature phenotype still induced apoptosis through a process dependent upon nitric oxide. However, nitric oxide itself was not directly toxic to motor neurons. To investigate whether intracellular superoxide contributed to nitric oxide-dependent apoptosis, we developed a novel method using pH-sensitive liposomes to deliver Cu, Zn superoxide dismutase intracellularly into motor neurons. Intracellular superoxide dismutase prevented motor neuron apoptosis from trophic factor withdrawal, whereas empty liposomes, inactivated superoxide dismutase in liposomes or extracellular superoxide dismutase did not. Neither hydrogen peroxide nor nitrite added separately or in combination affected motor neuron survival. Our results suggest that a partial reduction in trophic support induced motor neuron apoptosis by a process requiring the endogenous production of both nitric oxide and superoxide, irrespective of the extent of motor neuron maturation in culture.     

Reactive Macrophages Increase Oxidative Stress and Alpha-Synuclein Nitration During Death of Dopaminergic Neuronal Cells in Co-Culture: Relevance to Parkinson’s Disease

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons and a substantial decrease in the neurotransmitter dopamine in the nigro-striatal region of the brain. Increased markers of oxidative stress, activated microglias and elevated levels of pro-inflammatory cytokines have been identified in the brains of patients with PD. Although the precise mechanism of loss of neurons in PD remains unclear, these findings suggest that microglial activation may contribute directly to loss of dopaminergic neurons in PD patients. In the present study, we tested the hypothesis that activated microglia induces nitric oxide-dependent oxidative stress which subsequently causes death of dopaminergic neuronal cells in culture. We employed lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) as a reactive microglial model and SH-SY5Y cells as a model for human dopaminergic neurons. LPS stimulation of macrophages led to increased production of nitric oxide in a time and dose dependent manner as well as subsequent generation of other reactive nitrogen species such as peroxynitrite anions. In co-culture conditions, reactive macrophages stimulated SH-SY5Y cell death characterized by increased peroxynitrite concentrations and nitration of alpha-synuclein within SH-SY5Y cells. Importantly 1400W, an inhibitor of the inducible nitric oxide synthase provided protection from cell death via decreasing the levels of nitrated alpha-synuclein. These results suggest that reactive microglias could induce oxidative stress in dopaminergic neurons and such oxidative stress may finally lead to nitration of alpha-synuclein and death of dopaminergic neurons in PD.     

Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice

Rho kinase (ROCK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROCK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. Our previous studies have demonstrated that Fasudil, a selective ROCK inhibitor, induced neuroprotection in vitro. Here we used an in vivo model of hypoxia/reoxygenation (H/R) injury to examine the neuroprotective effect of Fasudil, and explore its possible mechanism(s) in vivo. H/R resulted in the loss of hippocampal neurons, accompanied by increased apoptosis of neurons in hippocampus. The expression of ROCK II and activity of ROCK in the brain were increased after H/R, and located only in microglia, but not in astrocytes and neurons. The administration of Fasudil inhibited the activity of ROCK in brain tissue and cultured microglia, and protected hippocampal neurons against H/R injury. Further immunohistochemical analysis and cytokine determination revealed that Fasudil inhibited inducible nitric oxide synthase immunoreactivity in microglia and pro-inflammatory factors in brain tissue after H/R, which is consistent with the observation wherein Fasudil reduced the pro-inflammatory factors nitric oxide, IL-1β, IL-6 and TNF-, and increased anti-inflammatory factor IL-10 in cultured microglia under normoxic or hypoxic conditions. Our results indicate that inhibition of ROCK by Fasudil may represent a useful therapeutic perspective by inhibiting microglial inflammatory responses in the CNS.     

Synergistic dopaminergic neurotoxicity of manganese and lipopolysaccharide: differential involvement of microglia and astroglia

Overexposure to manganese is known to cause damage to basal ganglial neurons and the development of movement abnormalities. Activation of microglia and astrocytes has increasingly been associated with the pathogenesis of a variety of neurological disorders. We have recently shown that microglial activation facilitates manganese chloride (MnCl₂, 10–300 μM)‐induced preferential degeneration of dopamine (DA) neurons. In this study, we report that combinations of MnCl₂ (1–30 μM) and endotoxin lipopolysaccharide (LPS, 0.5–2 ng/mL), at minimally effective concentrations when used alone, induced synergistic and preferential damage to DA neurons in rat primary neuron‐glia cultures. Mechanistically, MnCl₂ significantly potentiated LPS‐induced release of tumor necrosis factor‐alpha and interleukin‐1 beta in microglia, but not in astroglia. MnCl₂ and LPS were more effective in inducing the formation of reactive oxygen species and nitric oxide in microglia than in astroglia. Furthermore, MnCl₂ and LPS‐induced free radical generation, cytokine release, and DA neurotoxicity was significantly attenuated by pre‐treatment with potential anti‐inflammatory agents minocycline and naloxone. These results demonstrate that the combination of manganese overexposure and neuroinflammation is preferentially deleterious to DA neurons. Moreover, these findings not only shed light on the understanding of manganese neurotoxicity but may also bear relevance to the potentially multifactorial etiology of Parkinson’s disease.     

Nitric oxide-mediated mitochondrial damage: A potential neuroprotective role for glutathione

In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II–III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, l-buthionine-[S,r]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II–III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-γ for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II–III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.     

Dopamine modifies oxygen consumption and mitochondrial membrane potential in striatal mitochondria

Dopamine is a neurotransmitter that has been related to mitochondrial dysfunction. In this study, striatal intact mitochondria and submitochondrial membranes were incubated with different dopamine concentrations, and changes on mitochondrial function, hydrogen peroxide, and nitric oxide production were evaluated. A 35% decrease in state 3 oxygen uptake (active respiration state) was found after 1 mM dopamine incubation. In addition, mitochondrial respiratory control significantly decreased, indicating mitochondrial dysfunction. High dopamine concentrations induced mitochondrial depolarization. Also, evaluation of hydrogen peroxide production by intact striatal mitochondria showed a significant increase after 0.5 and 1 mM dopamine incubation. Incubation with 0.5 and 1 mM dopamine increased nitric oxide production in submitochondrial membranes by 28 and 49%, respectively, as compared with control values. This study provides evidence that high dopamine concentrations induce striatal mitochondrial dysfunction through a decrease in mitochondrial respiratory control and loss of membrane potential, probably mediated by free radical production.     

ERK1/2 and p38 mitogen-activated protein kinase mediate iNOS-induced spinal neuron degeneration after acute traumatic spinal cord injury

The enhanced production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of neuronal apoptosis after acute traumatic spinal cord injury (SCI). In the present study, to further characterize the pathways mediating the synthesis and release of NO, we examined activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) in microglia/macrophages in the injured area of adult rats subjected to a complete transection at the T10 vertebrae level and assessed their role in NO production and survival of neurons by using immunohistochemistry, Western blot, RT-PCR and pharmacological interventions. Results showed activation of microglia/macrophages featured by morphological changes, as visualized immunohistochemically with the marker OX-42, in the areas adjacent to the lesion epicenter 1 h after surgery. Concomitantly, iNOS mRNA and its protein in the activated microglia/macrophages were also significantly upregulated at early hours after surgery. Their levels were maximal at 6 h, persisted for at least 24 h, and returned to basal level 72 h after SCI. Furthermore, phosphorylated ERK1/2 and p38 MAPK were activated as well in microglia/macrophages in injured area with a similar time course as iNOS. With administration of L-NAME, a NOS inhibitor, the number of apoptotic neurons was clearly decreased, as assessed with TUNEL method at 24 h after SCI. In parallel, loss of neurons induced by SCI, assessed with NeuN immunohistochemistry, was also diminished. Moreover, the effect of inhibition of phosphorylation ERK1/2 and p38 MAPK by corresponding inhibitors PD98059 and SB203580 administered before and after SCI was also investigated. Inhibition of p38 effectively reduced iNOS mRNA expression and rescued neurons from apoptosis and death in the area adjacent to the lesion epicenter; whereas the inhibition of ERK1/2 had a smaller effect on decrease of iNOS mRNA and no long-term protective effect on cell loss. These results indicate the ERK1/2 and p38 MAPK signaling pathway, especially the latter, play an important role in NO-mediated degeneration of neuron in the spinal cord following SCI. Strategies directed to blocking the initiation of this cascade prove to be beneficial for the treatment of acute SCI.     

Developmental changes in the response of murine cerebellar granule cells to nitric oxide

Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.     

Ganoderma lucidum extract protects dopaminergic neurons through inhibiting the production of inflammatory mediators by activated microglia

Abundant evidence has suggested that neuroinflammation participates in the pathogenesis of Parkinson's disease (PD). The emerging evidence has supported that microglia may play key roles in the progressive neurodegeneration in PD and might be a promising therapeutic target. Ganoderma lucidum (GL), a traditional Chinese medicinal herb, has been shown potential neuroprotective effect in our clinical trials that lead us to speculate that it might possess potent anti-inflammatory and immunomodulating properties. To test this hypothesis, the present study investigated the potential neuroprotective effect of GL and underlying mechanism through inhibiting microglial activation using co-cultures of dopaminergic neurons and microglia. The cultures of microglia or MES23.5 cells alone or together were treated for 24 h with lipopolysaccharide (LPS, 0.25 μg/mL) as a positive control, GL extracts (50-400 μg/mL) or MES23.5 cell membrane fragments (150 μg/mL) were used in treatment groups. Microglia activation, microglia-derived harmful factors and [(3)H]dopamine ([(3)H]DA) uptake of MES23.5 cells were analyzed. The results showed that microglia were activated by LPS and MPP(+)-treated MES23.5 cell membrane fragments, respectively. Meanwhile, GL extracts significantly prevented the production of microglia-derived proinflammatory and cytotoxic factors, including nitric oxide, tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β), in a dose-dependent manner and down-regulated the TNF-α and IL-1β expressions on mRNA level. In addition, GL extracts antagonized the reduction of [(3)H]DA uptake induced by MPP(+) and microglial activation. In conclusion, these results suggest that GL may be a promising agent for the treatment of PD through anti-inflammation.     

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti‐inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV‐2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS‐induced M1 BV‐2 microglia, also the inflammatory secretion phenotype of M1 BV‐2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK‐/mTOR‐mediated autophagic flux stimulation.     

Nitric oxide is toxic to melanocytes in vitro

Nitric oxide is a diffusible gaseous mediator generated from l-arginine by inducible and constitutive nitric oxide synthases. It has been associated with cytotoxic effects. Inflammatory cells and Langerhans cells can express the inducible form of nitric oxide synthase and produce large quantities of nitric oxide. The proximity of these cells to melanocytes could result in melanocyte cell death. We studied melanocyte susceptibility to nitric oxide using the nitric oxide donor compound sodium nitroprusside and nitric oxide released by the Langerhans like cell-line XS-52 following stimulation with lipopolysaccharide (LPS). Melanocyte lysis, quantified by chromium release in the presence of sodium nitroprusside was both time and concentration dependent. Co-culture of LPS-stimulated XS cells with melanocytes also resulted in melanocyte cell death. No cell death was observed when melanocytes alone were exposed to LPS. Melanocytes were killed even when the co-cultures were performed across Transwells in which there was no direct contact between XS cells and melanocytes. XS-induced melanocyte death was thus dependent on a diffusible factor consistent with nitric oxide. Cell death was markedly decreased in co-cultures performed in the presence of hemoglobin, a nitric oxide quencher. The possible role that nitric oxide may play in disorders associated with loss of pigmentation is discussed.     

Induction of cytosolic NADPH-diaphorase/nitric oxide synthase in reactive microglia/macrophages after quinolinic acid lesions in the rat striatum: an electron and light microscopical study

Induction of nitric oxide synthase and increased production of nitric oxide in microglia may play a crucial role in neuronal damage and neurodegenerative disorders. In the present study we have used light and electron microscopical NADPH-diaphorase histochemistry as the visualization procedure for nitric oxide synthase to investigate the time-course and subcellular patterns of NADPH-diaphorase expression in microglia/macrophages of quinolinic acid-lesioned rat striatum. For light microscopy, NADPH-diaphorase histochemistry sections were stained with nitroblue tetrazolium, while for ultrastructural analysis the tetrazolium salt 2-(2-benzothiazolyl)-5-styryl-3(4-phthalhydrazidyl) tetrazolium chloride (BSPT) was applied. Light microscopical inspection revealed a progressively increasing number of positive cells with increasing intensity of NADPH-diaphorase staining in microglia/macrophages from day 1 after quinolinic acid injection onward. Electron microscopical examination revealed a membrane bound NADPH-diaphorase in quiescent microglia as well as in activated microglia/macrophages through all stages of the lesion studied. Predominantly membranes of the nuclear envelope and the endoplasmic reticulum were labeled with BSPT-formazan, while in advanced stages selective membrane portions of mitochondria, Golgi apparatus and plasmalemma were also stained. From day 5 onward after lesion induction, a very distinctive type of NADPH-diaphorase was observed, forming accumulations of electron-dense grains that were distributed differentially throughout cytoplasmic areas and phagocytic vacuoles. Dynamics of expression, unique cytosolic localization and occurrence exclusively in activated microglia/macrophages suggest that this particular NADPH-diaphorase activity probably reflects the inducible isoform of nitric oxide synthase, whereas the membrane-bound precipitate may represent the neuronal and/or the endothelial isoform of the enzyme.     

Involvement of heparanase in migration of microglial cells

Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature.     

Nitric Oxide Has Differential Effects on Currents in Different Subsets of Manduca sexta Antennal Lobe Neurons

Nitric oxide has been shown to regulate many biological systems including olfaction. In the moth olfactory system nitric oxide is produced in the antennal lobe in response to odor stimulation and has complex effects on the activity of both projection neurons and local interneurons. To examine the cell autonomous effects of nitric oxide on these cells, we used patch-clamp recording in conjunction with pharmacological manipulation of nitric oxide to test the hypothesis that nitric oxide differentially regulates the channel properties of these different antennal lobe neuron subsets. We found that nitric oxide caused increasing inward currents in a subset of projection neurons while the effects on local neurons were variable but consistent within identifiable morphological subtypes.     

β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

目的：建立β淀粉样肽（Aβ1-40）诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法：用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子仪;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Western blot检测刺激后海马神经元内诱导型一氧化氮合酶（iNOS）和硝基酪氨酸（NT）的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3（caspase-3）活性来评价神经元的损伤程度。结果：终浓度为10μmol／L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24-72h,海马神经元较对照组形态有明显变化;经Western blot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论：小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。     

Nitrated lipids decompose to nitric oxide and lipid radicals and cause vasorelaxation

Nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite have been receiving increasing attention as mediators of nitric oxide toxicity. Indeed, nitrated and nitrosated compounds have been detected in biological fluids and tissues of healthy subjects and in higher yields in patients under inflammatory or infectious conditions as a consequence of nitric oxide overproduction. Among them, nitrated lipids have been detected in vivo. Here, we confirmed and extended previous studies by demonstrating that nitrolinoleate, chlolesteryl nitrolinoleate, and nitrohydroxylinoleate induce vasorelaxation in a concentration-dependent manner while releasing nitric oxide that was characterized by chemiluminescence-and EPR-based methodologies. As we first show here, diffusible nitric oxide production is likely to occur by isomerization of the nitrated lipids to the corresponding nitrite derivatives that decay through homolysis and/or metal ion/ascorbate-assisted reduction. The homolytic mechanism was supported by EPR spin-trapping studies with 3,5-dibromo-4-nitrosobenzenesulfonic acid that trapped a lipid-derived radical during nitrolinoleate decomposition. In addition to provide a mechanism to explain nitric oxide production from nitrated lipids, the results support their role as endogenous sources of nitric oxide that may play a role in endothelium-independent vasorelaxation.