Abstract: Deletion of the p66(Shc) gene in mice results in reduced levels of oxidative stress and longer lifespan. Reactive oxygen species (ROS) can lead to tissue damage, particularly in the brain. In this study we extended previous findings on the behavioral phenotype of the p66(Shc-/-) mice. Cognitive performance of adult and old p66(Shc-/-) and p66(Shc+/+) mice was tested in a Morris water maze (MWM) task while general reactivity and pain sensitivity were assayed at adulthood, respectively, in an open field and by means of a tail flick test. Levels of brain-derived neurotrophic factor (BDNF), a neurotrophin involved in several aspects of synaptic plasticity, emotionality and pain sensitivity, were assessed in selected brain areas. P66(Shc-/-) adult subjects, compared to WT, overall showed a better performance in the MWM, lower emotionality and a higher pain threshold, in addition to increased basal levels of BDNF in the hippocampus, as well as decreased levels of oxidative stress markers in the same brain area. Although all aged subjects failed to learn the cognitive task, aged p66(Shc-/-) mice were characterized by a better physical performance. These results suggest an interaction between the p66(Shc) gene and specific signaling pathways involved in behavioral adaptation to stress and aging.
Abstract: ABSTRACT: Inhibition of adenosine A2A receptors (A2ARs) is neuroprotective in several experimental models of striatal diseases. However, the mechanisms elicited by A2AR blockade are only partially known, and critical aspects about the potential beneficial effects of A2AR antagonism in models of neurodegeneration still await elucidation. In the present study, we analyzed the influence of the selective A2AR antagonist SCH 58261 in a rat model of striatal excitotoxicity obtained by unilateral intrastriatal injection of quinolinic acid (QA). We found that SCH 58261 differently affected the expression of cyclooxygenase-2 (COX-2) induced by QA in cortex and striatum. The antagonist enhanced COX-2 expression in cortical neurons and prevented it in striatal microglia-like cells. Similarly, SCH 58261 differently regulated astrogliosis and microglial activation in the 2 brain regions. In addition, the A2AR antagonist prevented the QA-induced increase in striatal brain-derived neurotrophic factor levels. Because COX-2 activity has been linked to excitotoxic processes and because brain-derived neurotrophic factor depletion has been observed in mouse models as well as in patients with Huntington disease, we suggest that the final outcome of A2AR blockade (namely neuroprotection vs neurodegeneration) is likely to depend on the balance among its various and region-specific effects.
Abstract: OBJECTIVE: To investigate how the mode of delivery affects the level oxidative stress in newborns. DESIGN AND METHODS: 15-F(2t)-isoprostane, as index of oxidative stress, was measured in umbilical cord plasma samples from 37 infants born after vaginal delivery or caesarian section, using specific immuno-affinity extraction and immunoassay. RESULTS: 15-F(2t)-isoprostane levels were higher in infants born after vaginal delivery (n=18) compared to those delivered by elective caesarian section (n=19). CONCLUSIONS: 15-F(2t)-isoprostane is a sensitive biomarker of fetal oxidative stress during labor.
Abstract: BACKGROUND: Oxidative stress is believed to be an early event and a key factor in Alzheimer's disease (AD) pathogenesis and progression. In spite of an intensive search for surrogate markers to monitor changes related to oxidative stress in the brain, there is as yet no consensus about which markers to use in clinical studies. The measurement of peripheral anti-oxidants is an alternative way of evaluating the involvement of oxidative stress in the course of the disease. Given the complexity of peripheral anti-oxidant defence, variations in the levels of individual anti-oxidant species may not fully reflect the overall capacity to fight oxidant conditions. We therefore chose to evaluate the total reductive capacity (herein defined as anti-oxidant capacity, AOC) in serum from control subjects and AD patients in order to study the association between peripheral anti-oxidant defence, cognitive impairment and patient survival. METHODS: We measured the levels of AOC in serum samples from 26 cognitively normal controls and 25 AD patients (12 post-mortem confirmed) who completed the Cambridge Cognitive Assessment. Cognitive decline was assessed in a subgroup of 19 patients who underwent a second cognitive assessment 2 years after the initial visit. RESULTS: Serum AOC levels were lower in AD patients than in controls and were correlated with their cognitive test scores, although AOC levels were unrelated to cognitive decline assessed two years later. On the other hand, AOC levels were predictive of the length of patients' survival, with higher levels giving longer survival. CONCLUSION: This study indicates that peripheral anti-oxidant defences are depleted in AD patients. The results suggest that serum AOC is a good index of the general health status and prognosis of patients but does not necessarily reflect the extent to which vulnerable neuronal populations are protected from oxidant processes. Further studies are required to establish whether peripheral AOC measurements may be useful in identifying asymptomatic individuals or those with early symptoms at high risk of developing significant cognitive impairment or dementia.
Abstract: Minocycline has been shown to be neuroprotective in various models of neurodegenerative diseases. However, its potential in Huntington's disease (HD) models characterized by calpain-dependent degeneration and inflammation has not been investigated. Here, we have tested minocycline in phenotypic models of HD using 3-nitropropionic acid (3NP) intoxication and quinolinic acid (QA) injections. In the 3NP rat model, where the development of striatal lesions involves calpain, we found that minocycline was not protective, although it attenuated the development of inflammation induced after the onset of striatal degeneration. The lack of minocycline activity on calpain-dependent cell death was also confirmed in vitro using primary striatal cells. Conversely, we found that minocycline reduced lesions and inflammation induced by QA. In cultured cells, minocycline protected against mutated huntingtin and staurosporine, stimulations known to promote caspase-dependent cell death. Altogether, these data suggested that, in HD, minocycline may counteract the development of caspase-dependent neurodegeneration, inflammation, but not calpain-dependent neuronal death.
Abstract: Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-isoprostane 15-F(2t)-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites--produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,--was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F(2t)-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.
Abstract: Isoprostanes are a family of prostaglandin-like compounds that are generated in vivo by free radical attack of esterified arachidonic acid and then released in free form in biological fluids. Since their discovery in 1990, they have been extensively used as biomarkers of lipid peroxidation and oxidative damage in an increasing number of human diseases. Few members of the isoprostane family are biologically active and could contribute to the functional consequences of oxidant injury. The present review summarises the current knowledge on formation and biological activities of these lipid peroxidation products, focusing on their role as valuable biomarkers to investigate the involvement of oxidative stress in the pathogenesis of infant and adult central nervous system diseases. In addition to isoprostanes, a new class of free radical-mediated peroxidation products, named neuroprostanes, is discussed. Neuroprostanes derive from peroxidation of docosahexaenoic acid, a polyunsatured fatty acid particularly abundant in neurons, and may represent a more selective index of brain oxidant injury than isoprostanes. In spite of some discrepancies in the results reported in different studies, isoprostane and neuroprostane levels in human biological fluids, as well as in experimental models of brain diseases, appear to be valuable indicators not only to monitor the occurrence and the causal role of oxidative stress in brain pathologies, but also for critical selection and evaluation of appropriate antioxidant therapies.
Abstract: To understand the basis of oligodendrocyte (OL) susceptibility to oxidative injury, purified rat OL cultures at different stages of maturation were exposed to nitric oxide (NO) donors with fast or slow kinetics of release and to tert-butyl-hydroperoxide, a membrane-permeant organic hydroperoxide. OL precursors (pre-OL) displayed the highest vulnerability to both oxygen or nitrogen reactive species, whereas mature OLs were uniquely vulnerable to long-lasting levels of NO. Cell death occurred by necrosis as well as apoptosis associated with increased caspase-3 activity and, only in the case of pre-OLs, with a decreased expression of the anti-apoptotic protein bcl-2. Pre-OLs were also more susceptible than mature OLs to lipid peroxidation, as measured by F2-isoprostane content in culture media. Finally, pre-OLs, but not mature OLs, expressed high levels of the mitochondrial scavenging enzyme Mn superoxide dismutase, suggesting that pre-OLs may efficiently convert anion superoxide into hydrogen peroxide and, paradoxically, be more predisposed than mature OLs to a toxic imbalance between hydrogen peroxide production and detoxification processes. These data suggest that susceptibility to lipid peroxidation, expression of the scavenging enzyme Mn superoxide dismutase and of the anti-apoptotic protein bcl-2, may contribute to the maturation-dependent vulnerability of OLs to oxidant injury.
Abstract: BACKGROUND: The evidence that inflammation plays a pivotal role in the pathophysiology of acute coronary syndromes prompted us to investigate the effects of glucocorticoid treatment on leukotriene (LT) C4 and thromboxane (TX) A2 biosynthesis in unstable angina. METHODS AND RESULTS: Urinary LTE4 and 11-dehydro-TXB2 were significantly higher in 12 patients with unstable angina than in 12 patients with stable angina and 12 patients with nonischemic chest pain. Furthermore, we randomized the unstable angina patients to receive intravenous 6-methylprednisolone (6-MP; 1 mg/kg BID for 2 days) or matching placebo and collected 12 consecutive 6-hour urine samples before and during the infusions. LTE4 excretion showed a time-dependent decrease in the 6-MP group but did not decrease during placebo. Furthermore, during myocardial ischemia, LTE4 was significantly higher before 6-MP infusion than during steroid therapy. In contrast, 11-dehydro-TXB2 did not differ significantly during 6-MP versus placebo. Myocardial ischemia elicited by stress test in the stable angina patients was not accompanied by any change in LTE4 and 11-dehydro-TXB2, thus ruling out a role of ischemia per se in the induction of increased eicosanoid production. CONCLUSIONS: Increased production of vasoactive LT and TX may occur in unstable angina despite conventional antithrombotic and antianginal treatment. Glucocorticoids can suppress LTC4 biosynthesis in the short term and may provide an interesting tool to explore the pathophysiological significance of inflammatory cell activation in this setting.
Abstract: Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) are neuroprotective, although the mechanisms underlying their beneficial effect remain largely unknown. Given their well-known adverse effects, which of the NSAIDs is the best for neurodegenerative disease management remains a matter of debate. Paracetamol is a widely used analgesic/antipyretic drug with low peripheral adverse effects, possibly related to its weak activity as inhibitor of peripheral cyclooxygenase (COX), the main target of NSAIDs. As microglia play an important role in CNS inflammation and pathogenesis of neurodegenerative diseases, we investigate the effect of paracetamol on rat microglial cultures. Although less potent than other NSAIDs, (indomethacin approximately NS-398 > flurbiprofen approximately piroxicam > paracetamol approximately acetylsalicylic acid), paracetamol completely inhibited the synthesis of prostaglandin E(2) (PGE(2)) in lipopolysaccharide-stimulated microglia, when used at concentrations comparable to therapeutic doses. The drug did not affect the expression of the enzymes involved in PGE(2) synthesis, i.e., COX-1, COX-2, and microsomal PGE synthase, or the release of the precursor arachidonic acid (AA). Paracetamol inhibited the conversion of exogenous AA, but not PGH(2), into PGE(2) indicating that the target of the drug is COX activity. Consistently, paracetamol inhibited with similar IC(50) the synthesis of PGF(2alpha) and thromboxane B(2), two other COX metabolites. Finally, none of the NSAIDs affected the productions of nitric oxide and tumor necrosis factor(alpha), two inflammatory mediators released by activated microglia. As paracetamol was reported to inhibit PG synthesis in peripheral macrophages with an IC(50) at least three orders of magnitude higher than in microglia, we suggest that this drug represents a good tool for treating brain inflammation without compromising peripheral PG synthesis.
Abstract: The concentration of the cyclooxygenase product prostaglandin E(2) was sixfold higher in CSF samples from 18 cases of variant Creutzfeldt-Jakob disease (CJD) than in a group of eight subjects with other noninflammatory neurologic diseases, and comparable to those found in a group of six patients affected by diseases with a known inflammatory component. This finding suggests that cyclooxygenase activity may have a role in variant CJD pathogenesis, as previously reported in sporadic CJD.
Abstract: Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE(2) and 8-epi-PGF(2alpha) following infusion of N-methyl-D-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE(2) and 8-epi-PGF(2alpha) basal levels. These effects were largely prevented by the specific cytosolic phospholipase A(2) (cPLA(2) ) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.
Abstract: Isoprostanes are a family of biologically active molecules recently characterized, which is emerging as a new class of specific and reliable markers of in vivo and ex vivo lipid peroxidation and oxidative damage. These molecules are stable, relatively abundant and easily detectable by sensitive and specific analytical methods. In the last years, the measurement of their levels in tissue homogenates or biological fluids has significantly improved our knowledge on the involvement of oxidative stress in several neurological diseases. Here we present evidence indicating that isoprostanes can be successfully used also to study the mechanisms involved in free radical brain damage following hypoxic-ischaemic or inflammatory conditions in newborns and preterm infants.
Abstract: Isoprostanes are prostaglandin-like compounds which are formed by free radical catalysed peroxidation of arachidonic acid esterified in membrane phospholipids. They are emerging as a new class of sensitive, specific and reliable markers of in vivo lipid peroxidation and oxidative damage. Since their initial description of in 1990, the rapid development of analytical methods for isoprostane measurement has allowed to overcome some of the pitfalls of the previous and most widely used methods of assessing free radical injury. Here, we summarise the current knowledge on these novel class lipid peroxidation products and the advantages of monitoring their formation to better define the involvement of oxidative stress in neurological diseases. Although the literature data are still not abundant, they indicate that in vivo or post mortem cerebrospinal fluid and brain tissue levels of isoprostane are increased in some diseases such as multiple sclerosis, Alzheimer's disease, Huntington's disease, and Creutzfeldt-Jakob disease.
Abstract: The levels of 2 arachidonic acid metabolites formed either by enzymatic activity of cyclooxygenase, i.e. prostaglandin E2 (PGE2), or by free radical-catalyzed peroxidation, i.e. F2-isoprostane 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), were measured in the CSF of subjects with sporadic and familial Creutzfeldt-Jakob disease (CJD) and in brain homogenates of scrapie-infected mice. The CSF levels of both metabolites were increased in sporadic CJD (n = 52) and familial CJD (n = 10) patients when compared with a group of patients with noninflammatory disorders. Similarly, PGE2 and 8-epi-PGF2alpha levels were higher in brain homogenates obtained from C57BL/6J mice infected with the ME7 scrapie strain than in brain homogenates from control animals. As PGE2 is 1 of the most abundant prostaglandins released during inflammation and 8-epi-PGF2alpha is a quantitative marker of lipid peroxidation, our results provide in vivo biochemical evidence for the occurrence of inflammation and oxidative stress in human and experimental transmissible spongiform encephalopathies (TSEs), a concept so far based mainly on histopathological and in vitro evidence. Interestingly, in sporadic CJD patients, high CSF levels of PGE2, but not 8-epi-PGF2alpha, correlated with short survival time, suggesting that the inflammatory response correlates with the clinical duration of disease.
Abstract: The catalytic activity of carbonic anhydrase (CA) contained in the glomus cells of mammalian carotid bodies has been determined in vitro by a potentiometric method. Experiments performed on whole rabbit carotid bodies have shown a very low variability, in terms of the overall CA activity, among organs belonging to different animals maintained in normoxic conditions. Repeated assays performed on each carotid body have shown a marked decrease of the overall CA activity after the first assay, thus suggesting the presence of at least two different forms of enzyme. Experiments performed on carotid bodies belonging to rabbits maintained in normal, hyperoxic and hypoxic conditions have shown that the overall CA activity follows the sequence: hypoxic > normoxic > hyperoxic, matching with the corresponding physiological activity of the carotid body.
Abstract: The aim of our study was to characterize a model of human prostaglandin endoperoxide synthase-2 (PGHS-2) expression allowing the assessment of pharmacological inhibition in vitro and ex vivo. Heparinized human whole blood samples were incubated with lipopolysaccharide (LPS, 0.1-50 micrograms/ml) for 0 to 24 hr at 37 degrees C. The contribution of platelet PGHS-1 was suppressed by either pretreating the subjects with aspirin (300 mg 48 hr before sampling) or adding aspirin (10 micrograms/ml) in vitro at time 0. PGE2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 micrograms/ml LPS, PGE2 production averaged 12.1 +/- 6.2 ng/ml (mean +/- S.D., n = 7). Cyclooxygenase activity increased in parallel with the mass of a monocyte protein doublet analyzed by Western blot using antibodies directed against the carboxyl-terminal portion of human PGHS-2. Dexamethasone (2 microM) inhibited LPS-induced PGE2 production by 96 +/- 4% (mean +/- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGH-2 and thrombin-stimulated platelet PGHS-1. IC50 values (microM) for inhibition of PGHS-1 and PGHS-2 were: indomethacin, 0.70 +/- 0.20 vs 0.36 +/- 0.10 (P < .05); S-indobufen, 0.64 +/- 0.22 vs. 14.9 +/- 8 (P < .05), R-indobufen, 38 +/- 18 vs. 230 +/- 68 (P < .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +/- 96 vs. 187 +/- 96.(ABSTRACT TRUNCATED AT 250 WORDS)
