Authored by Chang-Quan Huang*
Abstract
Objective: this study observes the changes of cognitive function, β-amyloid peptide (Aβ) and Brain-derived growth factor (BDNF) in circadian rhythm disturbances (CRDs).
Method: Animal models of CRDs induced by light-dark cycles in different periods. Cognitive function in mice was detected by water maze test. BDNF and Aβ levels were determined ELISA Kit.
Result:
In CRDs, there were cognitive function declines, BDNF decreased and Aβ increased.Conclusion: CRDs can lead to cognitive function declines; it was related to BDNF decreased and Aβ increased.
Keywords: circadian rhythm disturbances; cognitive function; Brain-derived growth factor; β-amyloid peptide
Introduction
Several studies have demonstrated that circadian rhythm disturbances (CRDs) can lead to cognitive function declines [1]. The morbidity and prevalence of dementia (including Alzheimer’s disease) in people with CRDs are higher than those in the general population [2-4]. Even CRDs can lead to shorter life expectancy [2- 4]. The biological effects of CRDs are mostly concentrated in clinical studies, mainly in the observation of phenomena, and research on relevant mechanisms has been few done [1,7]. Improving CRDs is the best way to significantly reduce its harm, however, improving CRDs is very difficult and the method is very limited. Therefore, it is very important to elucidate the mechanism of the effects of CRDs on organisms.
It has been documented that Brain-derived growth factor (BDNF) expression is reduced in the brains of Alzheimer’s disease (AD) [8-13]. Patients AD are characterized by the accumulation of β-amyloid peptide (Aβ) within the hippocampus along with hyperphosphorylated [8-13]. Aβ level is decreased by BDNF in primary neuronal cultures, whereas it is elevated by BDNF deprivation [14,15]. BDNF may have a protective role against AD pathogenesis. The beneficial effect of BDNF administration has been shown to increase cognitive function in impaired animals, and studies in AD models show that BDNF has a neuroprotective effect against Aβ toxicity [16-18]. Neuronal cell growth is modulated by factors such as BDNF [19-21]. BDNF is highly concentrated in the hippocampus, important in synaptic plasticity, and contributes to neurogenesis in the dentate gyrus16-18. BDNF plays a pivotal role in age-related memory impairments and is associated with agerelated atrophy of the hippocampus [16-18]. Previous studies have reported that serum BDNF levels are reduced in AD [17,19]. A study of neuronal cell cultures found that amyloid peptide at sublethal concentrations interfered with neuronal plasticity mediated by BDNF signaling cascade [22].
The mechanisms underlying cognitive function declines or AD disease caused by CRDs has been unclear2. In individuals with CRDs disorders, BDNF decreases or Aβ increases, it’s only a hypothesis [2,7]. If this hypothesis can be confirmed, this may be a mechanism for cognitive impairment and increased Alzheimer’s disease caused by CRDs. In this study, we tested the BDNF and Aβ content in hippocampus of experimental animal models of CRDs to verify this hypothesis.
Experiment
Experimental animal and establishment of circadian rhythm model
90 C57BL/6 Male mice (Animal experiment center of Sichuan University, Chengdu, China), weighing 20-22 g (8 weeks old), were used in the experiments. The mice were maintained under standard laboratory conditions, and were housed in acrylic cages (20 cm 27 cm 12 cm) under an artificial 12-hour light/ dark cycle at a controlled temperature (22 ± 2°C) and humidity (60±10%), with free access to water and food unless specified otherwise departmental room for 7 days to ensure adaptation to the new environment. All animal procedures were conducted under the regulations of the Sichuan University Animal Care and Use Committee, Chengdu, China. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Light-dark cycle was as a method of studying the circadian rhythm [23]. Firstly, the mice were randomly divided into three groups: Light-dark cycle 12/12 hour group, Light-dark cycle 3/3 hour group and Light-dark cycle 22/22 hour group, each group included 30 mice. Light-dark cycle 12/12 hour group was as sham control, the other groups were as CRDs group. After grouping, mice in each group were fed for 12 weeks.
Cognitive function analysis
Water maze experiment was used to test cognitive function [24]. The water-maze tank was placed with four external visual cues and filled with water containing a nontoxic white color. The temperature of the water was maintained by 23–25°C. A white platform (10 cm in diameter and 30 cm in height) was placed in one of the quadrants with equal area and submerged 2 cm below the water surface. During each trial session, the escape latency time spent to find the hidden platform was monitored by a video tracking system. During the four subsequent days of training the mice were given three trials per day with the submerged platform in the pool. When the mouse located on the plated platform, it was allowed to remain on it for an additional 10 sec. If the mouse did not find the hidden platform within 120 sec, the mouse was guided to the platform and permitted to remain on it for an additional 10 sec. On the last day, the hidden platform was removed from the watermaze tank and probe test was performed. Mice were allowed to swim for 90 sec and the staying time in the maze quadrant where the platform had previously been located was recorded.
BDNF and Aβ analysis
BDNF and Aβ levels were determined as previously described using the mice BDNF and Aβ ELISA Kit (Thermo Fisher Scientific) [25]. Briefly, from three subregions of the hippocampus formation (CA1, CA3, and dentate gyrus), sample tissue was preservative and treated according to ELISA Kit instructions. As the tissue punches contained less tissue than the entire hippocampus, it was homogenized in 250μl of Lysis buffer (Promega) to prevent dilution and the full 100μl of sample specified in the kit was added to each well. As tissue weight could not be obtained, total protein levels were measured by Bradford assay. BDNF and Aβ concentration is expressed as ng/g protein.
Data analysis
Values from detected Morris Water Maze Tests, BDNF and Aβ level are reported as means ± SE. Statistical analyses include ANOVA (Comparison among the three groups) and Bonferroni t-tests (The comparison between the two groups). The overall level of statistical significance was p<0.05.
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