Tahereh Tabatabaie

Chapter 13 Nitrone-Based Free Radical Traps as Neuroprotective Agents in Cerebral Ischaemia and Other Pathologies

Nitrone-based spin trapping compounds have been shown to protect experimental animals from pathology associated with ischaemia/reperfusion injury, endotoxaemia, natural and accelerated aging, certain xenobiotics, and physical trauma. Moreover, these compounds have an intriguing nootropic action. Nitrones affect pathophysiological correlates in both the central nervous system and peripheral organ systems. These compounds have been shown to affect cellular oxidation state and oxidatively sensitive enzyme systems, but the precise mode of nitrone action has not been elucidated. Recent discoveries regarding the ability of nitrones to suppress gene transcriptional events associated with pathophysiological states, particularly the elaboration of NF kappa B-regulated cytokines and inducible nitric oxide synthase, argue that nitrones may act at a proximal level to oxidatively sensitive signal amplification systems.

Spin trapping agent phenyl N-tert-butylnitrone protects against the onset of drug-induced insulin-dependent diabetes mellitus

Insulin‐dependent diabetes mellitus is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the β‐cells. Cytokines and nitric oxide (NO) have been shown to be involved in this destruction. Phenyl N‐tert‐butylnitrone (PBN) has demonstrated protective effects against several pathological conditions including ischemia‐reperfusion injury and endotoxin‐induced shock. We report here that PBN co‐administration can prevent the onset of the STZ‐induced diabetes in mice. PBN co‐treatment inhibited the streptozotocin (STZ)‐induced hyperglycemia, the elevation in the level of glycated hemoglobin and weight loss in the treated mice. Histological observations indicated destruction of β‐cells in the STZ‐treated animals and its prevention by PBN co‐treatment. EPR spin trapping experiments in the pancreas indicated the in vivo formation of NO in STZ‐treated animals and its attenuation by PBN treatment.

Antioxidant amplifies antibiotic protection in the cecal ligation and puncture model of microbial sepsis through interleukin-10 production

Preadministration of antioxidants such as pyrrolidine dithiocarbamate (PDTC) and phenyl N-tert-butyl nitrone (PBN) protects animals from lethality in sepsis models. However, the requirement of preadministration greatly diminishes the clinical significance of these studies. Although the synthetic antioxidant PBN has been shown to effectively protect rodents from lethality in endotoxemia (lipopolysaccharide [LPS] model), preliminary screening indicates that pre- or postadministration of PBN does not protect in the rat cecal ligation and puncture (CLP) model. We show in this report that in a rat CLP model, the administration of PBN (150 mg/kg, 30 min after CLP) followed by the antibiotic imipenem (IMP; 10 mg/kg, 1 h after CLP) significantly increased survival compared with other single treatment groups. Previously, we have shown that PBNs protection in a rat LPS model is mediated by the overproduction of the anti-inflammatory cytokine interleukin (IL)-10. We show in this study that the increase in survival found in the PBN + IMP-treated group was abrogated by immunoneutralization with anti-IL-10 antibody, indicating that endogenous IL-10 is an effective protective factor. Plasma LPS levels were shown to be elevated after imipenem treatment, and the increased LPS level could have assisted to overproduce endogenous IL-10, as in the case of the PBN-treated LPS model. Statistical analysis indicated that the increase of IL-10 in PBN + IMP-treated group at early time period has significant association to the improvement of survival.

Interleukin-10 overexpression mediates phenyl-N-tert-butyl nitrone protection from endotoxemia

The free radical trapping compound phenyl N-tert-butylnitrone (PBN) provides potent protection against lethal endotoxemia in rodents, but the mechanism of this protection is not well understood. The objective of this study was to show that PBN administration in lipopolysaccharide- (LPS) induced endotoxemia promotes enhanced production of endogenous interleukin 10 (IL-10), and the expressed IL-10 is a causal factor in the protection from endotoxemia. We show the amplified expression of IL-10 in liver and plasma in PBN- (150 mg/kg) plus LPS- (4 mg/kg) treated rats using ribonuclease protection assay (RPA) and ELISA. In situ hybridization was utilized to visualize the overexpression of the IL-10 gene, and ELISA was used to determine plasma IL-10 and TNF&agr; levels. Plasma IL-10 showed a 3-fold increase in PBN/LPS-treated rats compared to those treated with LPS alone, and in contrast, TNF&agr; level decreased by more than 90%. However, the administration of PBN alone induced no IL-10 production. Immunoneutralization of IL-10 through anti-IL-10 antibody administration to PBN/LPS-treated rats abrogated PBNs suppression of systemic nitric oxide (NO) formation, a surrogate marker for the severity of endotoxemia, indicating that IL-10 is a causal factor for the protection. In these experiments, systemic NO level was quantified using an in vivo electron paramagnetic resonance (EPR) NO-trapping technique. Gel-shift and immunohistochemical analyses indicated that the transcription factor NF-&kgr;B was deactivated after PBN treatment, suggesting that NF-&kgr;B deactivation is closely involved in IL-10 overexpression.

Reactive Oxygen Involvement in Neurodegenerative Pathways

As organs age, the likelihood of severe dysfunction increases steadily. The brain is particularly sensitive to age-related, chronic and acute oxidative pathologies. An emerging paradigm holds that diverse neurodegenerative conditions share a common etiological factor, namely, enhanced brain tissue oxidation owing to exacerbated production of reactive oxygen species (ROS) or to compromise of antioxidant defense and repair mechanisms. Brain is particularly susceptible to oxidative stress owing to its high content of unsaturated lipids, high metabolic rate, relative dearth of antioxidant enzymes, and inability to regenerate lost neurons. Pathogenic ROS generation may result from metabolic enzyme dysregulation, impaired mitochondrial respiration, excitotoxic stimulation, and secondarily as a function of intracellular calcium stress (summarized in Fig. 1 and elaborated below). Natural variation in antioxidant systems may explain why humans differ so greatly with respect to pathways and rates of neurodegeneration. If this is the case, antioxidant supplementation of the aging brain may forestall certain aspects of age-related neurodegeneration. Accordingly, much research has focused on antioxidant management of aging brain and on antioxidant interdiction of postischemic brain damage. Recent findings indicate that specific antioxidants do more than scavenge ROS, but may indirectly affect cellular signal transduction, genetic response, and inflammatory events in such a way as to modulate beneficially brain response to oxidative challenge.

Direct Administration of Interleukin-1 and Interferon-γ to Rat Pancreas Leads to the In Vivo Production of Nitric Oxide and Expression of Inducible Nitric Oxide Synthase and Inducible Cyclooxygenase

Introduction Proinflammatory cytokines may play a pivotal role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In vitro, the formation of nitric oxide (NO) catalyzed by inducible NO synthase (iNOS) has been shown to be involved in the cytotoxic effects of cytokines on pancreatic β cells. Cytokines have also been shown to cause the expression of inducible cyclooxygenase (COX-2) in isolated islets. Aims To describe a novel in vivo model that allows investigation of the effects of direct cytokine administration to the pancreas. Methodology and Results By using this method, we demonstrate that administration of interleukin-1β and interferon-&ggr; to rat pancreas results in the generation of NO in the treated pancreata as detected by NO trapping and electron paramagnetic resonance spectroscopy. β cells were identified as the source of the formed NO. Reverse transcription and polymerase chain reaction analyses showed that administration of cytokines to the pancreas leads to the expression of iNOS and COX-2 mRNA in the pancreas tissue as well as the islets isolated from such tissues. The compound phenyl N-tert-butylnitrone, which protects mice against streptozotocin-induced IDDM, inhibits NO formation and downregulates both iNOS and COX-2 mRNA levels.