Introduction Oxidative Stress Has Been Involved in the Mecha- Nisms of Biologic Aging, as Well as in the Pathogen- Esis

OXIDATIVE STRESS HAS BEEN INVOLVED IN THE MECHANISMS OF BIOLOGIC AGING, AS WELL AS IN THE PATHOGENESIS OF CANCER, ATHEROSCLEROSIS, DIABETES, AND NEURODEGENERATIVE DISORDERS.1 Oxidative stress occurs whenever there is an imbalance between oxidant production and antioxidant defenses, either because the former is increased, because the latter are decreased, or both. At the cellular level, such imbalance can result in structural damage due to oxidative modifications of proteins, lipids, and nucleic acids. Major cellular oxidants include reactive oxygen species (ROS, eg, O2and H2O2) and reactive nitrogen species (RNS, eg, NO). Although most ROS are the byproducts of the electron transport chain, oxidants can also be produced by extramitochondrial sources such as NADPH oxidases and nitric-oxide synthases.1,2 Several hypotheses about the functions of sleep rest on the assumption that wakefulness represents an oxidative challenge for the brain. It has been claimed, for instance, that sleep may allow the removal of free radicals accumulated in the brain during wakefulness.3 Moreover, it has been proposed that, during sleep, uridine and glutathione may facilitate the oxidative detoxification of the brain by potentiating GABAergic transmission and inhibiting glutamatergic transmission, respectively.4 Consistent with these hypotheses, brain energy metabolism, which relies almost completely on mitochondrial respiration, is higher in wakefulness than in non-rapid eye movement (NREM) sleep.5 Moreover, peripheral metabolic rate,6 cerebral cortex glutamatergic transmission,7 and extracellular nitric-oxide concentration8,9 are also increased in spontaneous wakefulness and/or sleep deprivation relative to NREM sleep. Whether sleep deprivation causes oxidative damage, however, remains unknown, nor it is clear why sleep could protect against oxidative stress. Brain energy metabolism is as high in wakefulness as in rapid eye movement (REM) sleep, which represents 10% to 30% of total sleep.5 Moreover, prolonged sleep deprivation in animals is accompanied by a decrease, rather than an increase, in cerebral glucose utilization10 and is followed by an earlier and marked rebound of REM rather than NREM sleep.11 A few studies have examined signs of oxidative stress after 4 days of sleep deprivation with the flowerpot technique, which produces relatively selective REM sleep deprivation. Only 1 study12 controlled for immobilization and isolation stress and found no evidence that REM sleep deprivation per se causes changes in lipid peroxidation or in antioxidant defenses. Later studies from the same authors found a decrease in glutathione levels in the hypothalamus and thalamus of experimental rats relative to controls,13,14 but the use of single, instead of multiple, platforms did not allow the authors to tease apart the effects of sleep loss from those of immobilization stress. Immobilization stress alone can induce several markers of oxidative damage in the brain and in peripheral tissues.15,16 Finally, Ramanathan et al17 recently found that rats deprived of total sleep for 2 weeks show a decrease in the hippocampal and brainstem activity of 1 antioxidant enzyme, Cu-Zn superoxide dismutase. These authors did not assess other antioxidant enzymatic activities, nor did they measure oxidant production or any other marker of oxidative damage. The aims of the present study were 4-fold: (1) measure antioxidant enzyme activity and cellular ROS-RNS production to determine whether sleep deprivation causes oxidative stress and whether recovery sleep after sleep loss relieves the oxidative stress, (2) measure lipid peroxidation and protein oxidation in search for direct evidence of cellular damage after sleep deprivation, (3) determine whether sleep deprivation has differential effects in the brain compared to peripheral tissues, and (4) compare the effects of short-term sleep deprivation (8 hours) with those of sustained sleep loss (3-14 days). Preliminary results of this study have been published in abstract form.18