Melatonin Successfully Rescues the Hippocampal Molecular Machinery and Enhances Anti-oxidative Activity Following Early-Life Sleep Deprivation Injury

Abstract

Early-life sleep deprivation (ESD) is a serious condition with severe cognitive sequelae. Considering hippocampus plays an essential role in cognitive regulation, the present study aims to determine whether melatonin, a neuroendocrine beard with significant anti-oxidative activity, would greatly depress the hippocampal oxidative stress, improves the molecular machinery, and consequently exerts the neuro-protective effects following ESD. Male weanling Wistar rats (postnatal day 21) were subjected to ESD for three weeks. During this period, the animals were administered normal saline or melatonin (10 mg/kg) via intraperitoneal injection between 09:00 and 09:30 daily. After three cycles of ESD, the animals were kept under normal sleep/wake cycle until they reached adulthood and were sacrificed. The results indicated that ESD causes long-term effects, such as impairment of ionic distribution, interruption of the expressions of neurotransmitters and receptors, decreases in the levels of several antioxidant enzymes, and impairment of several signaling pathways, which contribute to neuronal death in hippocampal regions. Melatonin administration during ESD prevented these effects. Quantitative evaluation of cells also revealed a higher number of neurons in the melatonin-treated animals when compared with the saline-treated animals. As the hippocampus is critical to cognitive activity, preserving or even improving the hippocampal molecular machinery by melatonin during ESD not only helps us to better understand the underlying mechanisms of ESD-induced neuronal dysfunction, but also the therapeutic use of melatonin to counteract ESD-induced neuronal deficiency.

Keywords: TOF-SIMS analysis; early-life sleep deprivation; hippocampus; melatonin; neurochemical expression.

Figure 1

The effects of melatonin on Na⁺/K⁺ ATPase function. Photomicrographs (A) and histogram (B) show the extent of Na⁺/K⁺ ATPase expressions (arrow) in the CA1 and CA3 regions of hippocampus. Note that in ESC group, numerous neurons with strong Na⁺/K⁺ ATPase expressions were detected in the hippocampus. However, following ESD, remarkable decrease in hippocampal Na⁺/K⁺ ATPase expression was detected. In ESD-M10 group, hippocampal Na⁺/K⁺ ATPase expressions were effectively preserved. The immunoblotting data (C,D) coincided well with IHC findings in that melatonin effectively improved Na⁺/K⁺ ATPase activity. Scale bar: 300 μm.

Figure 2

Melatonin restores the trans-membrane ionic gradient following ESD injury. TOF-SIMS positive spectra show the hippocampal Na⁺ and K⁺ expressions in the ESC group (A,D), ESD group (B,E), and ESD-M10 group (C,F). Note that in ESD rats, increases in hippocampal Na⁺ expression were more significant than in ESC and ESD-M10 rats (p < 0.05), but K⁺ expression was significantly reduced when compared with the other two groups (p < 0.05). Paraformaldehyde was used for mass calibration with a set of standard peaks to improve the effect of ion spectra on the substrate (G–I).

Figure 3

Photomicrograph (A), schematic diagram (B), positive ion images (C–K), and histograms (L,M) showed both Na⁺ and K⁺ expression in the hippocampus of ESC (C,F,I), ESD (D,G,J), and ESD with melatonin treatment (E,H,K) groups. Note that in ESC group, most of the hippocampal neurons (arrows in C) were devoid of Na⁺ signal in the intracellular portions. On the contrary, intense K⁺ signal was kept in the intracellular part of hippocampal neurons (arrows in F). However, following ESD, strong Na⁺ signal was accumulated in the intracellular portion of some hippocampal neurons (arrows in D), suggesting an impairment of Na⁺ pump was resulted from ESD. Nevertheless, after ESD and treated with melatonin, the distribution pattern of both Na⁺ and K⁺ was restored to nearly normal profile in which no significant Na⁺ signal was detected in the intracellular portion of hippocampal neurons (arrows in E). Quantitative data of the spectral intensity corresponded well the imaging findings (L,M). The blue box in (B) showed the detecting region of the ionic image. Scale bars: 50 μm.

Figure 4

Photomicrographs (A–C) and histogram (D) show the extent of GABA expressions in the CA1 region of the hippocampus (A–C). Note that ESD rats demonstrated decreased GABAergic interneuron density in the hippocampus. In ESD-M10 group, the hippocampal GABA immune-expression significantly increased (C). Immunoblots (E) and histograms (F,G) show the protein expression level of 5-HT1 A and NMDAR in the hippocampus of ESC, ESD, and ESD-M10 groups. These activities were highest among ESC rats. Following ESD, protein expressions involved in neurotransmitter and receptor activities were significantly reduced (p < 0.05). However, in the animals treated with melatonin during the ESD period, effective expressions of 5-HT1 A and NMDAR significantly increased when compared with the ESD group (p < 0.05). Scale bars: 200 μm.

Figure 5

Photomicrographs (A,B) and histogram (C,D) show the extent of CREB and p-CREB activities in the CA1 and CA3 region of the hippocampus. Note that the protein expression levels of p-CREB markedly increased in the ESC and ESD-M10 groups when compared with the ESD group (p < 0.05). There was no difference between the three groups in terms of CREB protein expression in the hippocampus. Scale bars: 300 μm.

Figure 6

Photomicrographs (A) and histogram (B) show SOD1 expressions in the CA1 and CA3 region of the hippocampus based on immunohistochemistry results. Note that ESD rats showed decreased SOD1 expression in the hippocampus. In the ESC and ESD-M10 groups, hippocampal scheme 1 immune-expression significantly increased (p < 0.05). Immunoblots (C) and histograms (D–H) show the expressions of Nrf2 (D), p-Nrf2 (E) and the downstream antioxidant enzymes including SOD1 (F), CAT (G), and GSH-Px (H) of ESD-M10 group which were higher than in ESD group (p < 0.05). Note that melatonin effectively promotes anti-oxidative enzyme activities. Scale bars: 300 μm.

Figure 7

Photomicrographs and histogram show the results of Nissl staining of hippocampal neurons. There were decreases in cell density in CA1 and CA3 following ESD. Staining intensity increased with melatonin treatment. The histogram shows the quantification of the hippocampal CA1 and CA3 neurons. Nissl stained hippocampal neurons significantly increased in ESC and ESD-M10 groups when compared with ESD group (p < 0.05). Scale bars: 100 μm.

Figure 8

Schematic diagram showed the potential mechanism(s) of early-life sleep deprivation (ESD) on the induction or development of neuronal deficiency. ESD would both impair the Na⁺/K⁺ ATPase and depress the Nrf2-mediated anti-oxidative enzymes activities that consequently lead to neuronal deficiency through enhanced oxidative stress and cellular bioenergetics disruption. Exogenous application of melatonin could successfully preserve the neuronal function through effectively rescuing the molecular machinery of the neurons and significantly increasing neuronal anti-oxidative activity.