| Sleep, a spontaneous and restorative physiological process, occurs periodically and widely throughout most mammals and birds. It is well documented that the restorative effect of sleep plays an essential role in many biological functions, such as energy metabolism, strengthen immunity, memory consolidation, attention and emotion.Accordingly, sleep disorders can seriously damage the quality of life including excessive sleepiness, cognitive deficits, increased risk of cardiovascular and endocrine disorder,accidents and all-cause mortality in epidemiological and laboratory studies. In polysomnographic studies, a normal night of sleep is characteristiced by 4 to 6 repeated cycles of two distinct phases: non-rapid eye movement(NREM) and rapid eye movement(REM), each lasting approximately 90 to 110 minutes. Although so far the molecular mechanism of sleep is not completely understood, most researchers believe that the current model of sleep regulation is composed of two principle mechanisms, the homeostatic process and the circadian process. The former determines the appropriate timing of sleep, and which is complemented by the latter, a sleep drive, homeostatically building up an increased need for sleep in response to extended wake periods. Thehypothalamus is considered to be sleep-wake regulation center in which main nuclei include suprachiasmatic nucleus(SCN) as master circadian pacemaker, sleep-active neurons located preoptic/anterior hypothalamus(POAH) and promoted awake neurons located posterior and lateral hypothalamus such as orexin/hypocretin nucleus and tuberomammillary nucleus(TMN), in addition to the basal forebrain(BF) and nucleus in the tegmentum and pons. In recent years, orexin/hypocretin nucleus in the ventral-posterolateral hypothalamus become the focus to explore the sleep-wake mechanism.The orexins, also known as hypocretins(Hcrts), consist of orexin A(a 33-amino acid peptides, OA) and orexin B(a 28-amino acid peptides, OB), cleaved from a single precursor(prepro-orexin) produced in the posterior lateral hypothalamus. Over the past decade, previous studies have identified the orexin signaling system widely distributed throughout the whole brain, to participate in multiple physiological functions, including sleep/arousal, energy homeostasis, neuroendocrine, reward system and stress reaction.And orexin/hypocretin deficiency was found in sleep disorders such as narcolepsy with cataplexy, obesity, anxiety disorder, aging and neurodegenerative diseases. The previous study is the first evidence of the loss of Orexin A~+ neurons in the hypothalamus of rat after single platform sleep deprivation(>24h), but the underlying mechanism is unclear.Recent studies have found that epigenetic mechanism of gene- environment interaction is likely to play an important role in sleep and sleep disorders, espically with in-depth research in histone protein post-translational modifications(PTMs), such as histone H3 lysine 9 acetylation(H3K9ac) and H3K14 ac resulting in chromatin remodeling to activate gene expression. However, whether SD affects the levels of histone acetylation in discrete sleep-related brain areas or not? Whether histone acetylation modification involves in the impairment of Orexin A~+ neurons after SD in the hypothalamus or not? At present has not been reported. Therefore, our study focuses on exploring the changes of histone acetylation modification in sleep-related brain areas, and further adopting an HDAC inhibitor SAHA to estimate the changes of Orexin A expression, to provide the theoreticalevidence and treatment direction for clinical sleep disorders.Objective(1)To study the effects of the modified multiple platform sleep deprivation on weight and behavior of adult rats;(2)to explore the changes of histone acetylation modification in sleep-related brain areas;(3)to observe whether histone acetylation affects sleep deprivation associated impairment of hypothalamic Orexin A neurons.Methods(1)A modified multiple platform method was used to establish SD model in this study. The change of the weight and anxiety behavioral state were examined by the weight record, real-time video monitoring, Elevated plus maze test and Open field test;(2)A modified multiple platform method was used to establish SD model in this study. The levels of histone H3K9/H3K14 site acetylation modification were observed by Western blot and immunofluorescence staining in sleep-related brain areas;( 3) A modified multiple platform method was used to establish SD model in this study. The expression of Orexin A~+ neuron, Orexin A/H3K9 Ac, Orexin A/H3K14 Ac, cleaved caspase-3 was observed by immunofluorescence staining. Real- time PCR was applied to detect the changes in the levels of other orexinal markers m RNA in the hypothalamus; then intraperitoneal injection of SAHA(25 mg/kg/d)or DMSO, immunofluorescent staining was used to visualize the Orexin A~+ neurons in the hypothalamus.Results(1)During the period of sleep deprivation, the anxiety behavior of across, support, rear,hang increased significantly in rats, however, the numbers of rear and hang began to rapidly reduce in SD4 d. such activities were to minimize in SD6 d, maybe associated with muscle fatigue. Elevated plus maze test showed the anxiety level obviously increased in rats after SD6 d, and Open field test similarly showed increasing trend. Rats weight increased slowly, or even less along sleep deprivation process.(2)At 1d, 3d or 6d after SD,the levels of acetylation modification of histone H3K9 or H3K14 were obviously declined compared with the control group in hypothalamus and hippocampus, but there were no significant differences among three time points. At 6d after SD, however, the levels of acetylation modification of histone H3K9 or H3K14 were obviously increased in medial prefrontal cortex and rephe nucleus, the other three groups had no significant difference.(3)Compared with control group, at 1 d, 3 d and 6 d after SD, the numbers of Orexin A~+neurons and the levels of other orexinal markers m RNA were significantly reduced,without increasing neuronal apoptosis in hypothalamus. Acetylated modification H3K14 and H3K9 widely express in the central nervous, including Orexin A~+ neurons. whereas an HDAC inhibitor SAHA(25mg/kg, i.p.) could partly attenuate SD-induced damage of Orexin A~+ neurons.Conclusion( 1) The modified multiple platform method(MMPM) sleep deprivation can significantly induce anxiety-like behaviors in adult rats; to avoid the interference of energy metabolization obstacle factors, the present study prompted the point of SD3 is recommended for the best time in the next step experiment;(2)After continuous SD, the changes of histone acetylation modification are not identical in sleep-related brain areas,among which local specific gene expression probably be activated or silence. It point out that histone acetylation modification may participate in the regulation of sleep/wake;(3)SD may reduce the number of Orexin A~+ neurons in rat hypothalamic neurons by down-regulating the levels of histone acetylation. |
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