Ž . Brain Research 840 1999 184–193 www.elsevier.comrlocaterbres Interactive report No evidence of brain cell degeneration after long-term sleep deprivation in rats 1 Chiara Cirelli a, ) , Paul J. Shaw a , Allan Rechtschaffen b , Giulio Tononi a a The Neurosciences Institute, 10640 John J. Hopkins DriÕe, San Diego, CA 92121, USA b Sleep Research Laboratory, The UniÕersity of Chicago, Chicago, IL, USA Accepted 29 June 1999 Abstract Sleep deprivation leads to cognitive impairments in humans and, if sustained for 2–3 weeks in rats, it is invariably fatal. It has been suggested that neural activity associated with waking, if it is not interrupted by periods of sleep, may damage brain cells through excitotoxic or oxidative mechanisms and eventually lead to cell death. To determine whether sustained waking causes brain cell degeneration, three parallel strategies were used. The presence and extent of DNA fragmentation was analyzed with the TUNEL Ž . technique on brain sections from rats sleep deprived for various periods of time from 8 h to 14 days and from their respective controls. Ž . Adjacent sections from the same animals were stained with a newly developed fluorochrome Fluoro-Jade specific for degenerating neurons. Finally, total RNA from the cerebral cortex of the same animals was used to determine whether the expression of several stress response genes and apoptosis-related genes is modified after sustained waking. In most long-term sleep deprived rats only a few scattered Ž . TUNEL positive nuclei 1–3 were found in any given brain section. The overall number, distribution, and morphology of TUNEL positive cells in long-term sleep deprived rats did not differ significantly from yoked controls, short-term sleep deprived rats, and sleep controls. No evidence of degenerating neurons as detected by Fluoro-Jade was found in any experimental group. mRNA levels of all the stress response genes and apoptosis-related genes tested did not differ between long-term sleep deprived rats and their yoked controls. These results argue against the hypothesis that sustained waking can significantly damage brain cells through excitotoxic or oxidative mechanisms and that massive cell death may explain the fatal consequences of sleep deprivation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; cDNA array; Cell death; Fluoro-Jade; Glutamate; TUNEL 1. Introduction The harmful effects of sleep deprivation have been wx recognized for some time 3 . In humans, short periods of sleep loss can result in significant cognitive deficits while long-term disturbances in sleep have been associated with w x reduced longevity 22,24 . In rats, prolonged sleep depriva- Ž . tion 2–3 weeks leads to a syndrome characterized by increased food intake, weight loss, increased energy ex- penditure, and progressive decline in body temperature w x which is invariably fatal 36,37 . Although the mechanisms whereby sleep loss produces these effects are unknown, several recent theories have proposed that the neural activ- ity associated with waking, if sustained for long periods, ) Corresponding author. Tel.: q1-619-626-2110; Fax: q1-619-626- 2199; E-mail: cirelli@nsi.edu 1 Published on the World Wide Web on 8 July 1999. may damage brain cells and eventually lead to cell death w x 23,29,38 . According to these theories, a key function of sleep would be to relieve the brain from the harmful cellular stress of waking. Current understanding of the cell death cascade in the brain emphasizes the role of excess glutamate, which can damage several cellular components by increasing the intracellular levels of Ca 2q and free radicals. Cell-damag- ing effects of glutamate can be potentiated by glucocorti- w x coids, catecholamines, and indolamines 28,39 . Further- more, even normal levels of glutamate may be neurotoxic w x when intracellular energy levels are reduced 30 . Brain levels of catecholamines and indolamines are higher in w x waking than in sleep 1,8,34 . Short-term sleep deprivation w x increases glucocorticoid levels 27 as well as cate- wx cholamine synthesis and metabolism 2 . Rats subjected to Ž . chronic total sleep deprivation TSD show elevated levels of plasma norepinephrine and epinephrine and an increase 0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0006-8993 99 01768-0