Abstract: BACKGROUND: Neuronal apoptosis increases after brain aging and the mechanism is not clear. Recent studies have shown that melatonin can regulate the expression of a variety of apoptosis factors and enriched environments have been used as an effective treatment for Alzheimer’s disease. However, whether the combination of the two has better efficacy and their respective mechanisms of action remain unclear.
OBJECTIVE: To investigate the effects of enriched environment combined with melatonin intervention on learning and memory function and brain neuron apoptosis in SAMP8 mice.
METHODS: Twenty-four 3-month-old male SAMP8 mice were randomly divided into control group, enriched environment (EE) group, melatonin (MT) group, and enriched environment combined with melatonin (EE+MT) group, with 6 mice in each group. Mice in the control and MT groups were fed in a standard environment and those in the EE and EE+MT groups were fed in an enriched environment until they were 4 months old. At the age of 4 months, mice in the MT and EE+MT groups were subcutaneously injected with melatonin (8 mg/kg/d) for 30 days and those in the control and EE groups were given the same amount of normal saline for 30 days. After treatments, all the animals were kept in the original environment until 11 months of age, and their learning and memory abilities were evaluated by nest building test and Morris water maze test. After the completion of behavioral experiments, the apoptosis of hippocampal CAI cells was observed by immunofluorescence/TUNEL double standard staining and Nissl staining. The expression levels of apoptosis-related proteins Caspase-3, Bax, Bcl-2, and p53 were detected by western blot assay.
RESULTS AND CONCLUSION: Compared with the control group, there was a significant increase in the score of the nest building test, cross-platform frequency, NeuN immunoreactivity in the hippocampal CA1 region, the number of Nissl-positive cells and the expression level of Bcl-2 protein in the hippocampus in the EE, MT, and EE+MT groups (P < 0.05). These indicators were significantly higher in the EE+MT group than the EE and MT groups (P < 0.05). Hematoxylin-eosin staining results indicated that compared with the control group, the EE, MT, and EE+MT groups showed better morphology and arrangement of nerve cells in the hippocampal CA1 region. Compared with the control group, Morris water maze escape latency, the number of immunofluorescence/TUNEL double positive cells in the hippocampal CA1 region and the expression levels of Caspase-3, Bax and p53 proteins in the hippocampus were significantly decreased in the EE, MT, and EE+MT groups (P < 0.05). These indicators in the EE+MT group were significantly lower than those in the EE and MT groups (P < 0.05). Overall, both enriched environment and melatonin can improve learning and memory function and brain neuron apoptosis in SAMP8 mice. The mechanisms may be related to the regulation of P53/P21 pathway in mouse hippocampal neurons to reduce apoptosis. Moreover, the combined application of enriched environment and melatonin is likely to be more effective than a single regimen treatment.

Key words: rapid aging mice, SAMP8 mouse, enriched environment, melatonin, neuronal apoptosis, neurological function