Abstract:

Objective To discuss the suitable concentration of D-galactose （D-gal） and modeling period， and establish its induced aging-related cognitive dysfunction model in the mice， and perform a comprehensive evaluation. Methods Fifty C57BL/6J mice were randomly divided into control group and 100， 200， 400， and 800 mg·kg^-1 D-gal groups， with 10 mice in each group. The mice in various D-gal groups were subcutaneously injected with the corresponding concentration of D-gal once daily； the mice in control group were injected with an equal volume of normal saline. The body mass and water consumption of the mice in various groups were monitored； forelimb grip strength test and experiment on the ability of pole climbing sports were used to evaluate the motor coordination ability of the mice in various groups； novel object recognition test， Y maze test， and Morris water maze test were used to evaluate the cognitive function of the mice in various groups； HE staining and Nissl staining were used to observe the pathomorphology of brain tissue of the mice in various groups； immunohistochemistry method was used to detect the expression of β-galactosidase （β-gal） protein in brain tissue of the mice in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the mRNA expression levels of interleukin （IL）-1β， tumor necrosis factor-α （TNF-α）， IL-18， and IL-4 in hippocampus tissue of the mice in various groups； Western blotting method was used to detect the expression levels of β-gal， p53， and p16 proteins in hippocampus tissue of the mice in various groups. Results The body mass growth trends of the mice in control group and various D-gal groups were consistent and there was no statistically significant difference （P>0.05）， and there was no statistically significant difference in water consumption （P>0.05）. After 8 weeks of subcutaneous injection of D-gal， compared with control group， the forelimb grip strength values of the mice in 200 and 400 mg·kg?1 D-gal groups were significantly decreased （P<0.05 or P<0.01）； the pole-climbing time of the mice in 200 mg·kg?1 D-gal group was significantly prolonged （P<0.05）； the recognition indexes of the mice in 200 and 400 mg·kg?1 D-gal groups were significantly decreased （P<0.01）； the spontaneous alternation rate of the mice in 100， 200， 400， and 800 mg·kg?1 D-gal group was significantly decreased （P<0.05 or P<0.01）， the escape latency was significantly increased （P<0.05）.Spatial probe test showed that compared with control group， the escape latency of the mice in 200 mg·kg?1 D-gal group was significantly increased （P<0.05）. The HE staining and Nissl staining results showed that compared with control group， the hippocampus neurons of the mice in 200 mg·kg^-1 D-gal group were arranged disorderly， with obvious nuclear pyknosis， nuclear condensation， and abnormal morphology and structure， and the number of Nissl staining positive cells was significantly decreased. The immunohistochemistry results showed that compared with control group， the β-gal expressions in CA1 region， CA3 region， and cortex region of hippocampus tissue of the mice in 200 mg·kg?1 D-gal group were strongly positive. The RT-qPCR results showed that compared with control group， the expression levels of IL-1β，IL-18， and TNF-α mRNA in hippocampus tissue of the mice in 200 mg·kg?1 D-gal group were significantly increased （P<0.05 or P<0.01）， and the expression level of IL-4 mRNA was significantly decreased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of β-gal， p53， and p16 proteins in hippocampus tissue of the mice in 200 mg·kg?1 D-gal group were significantly increased （P<0.05 or P<0.01）. Conclusion The aging-related cognitive dysfunction model in the mice can be established by subcutaneous injection of 200 mg·kg?1 D-gal daily for 8 weeks.

Key words: Aging, D-galactose, Cognitive dysfunction, Disease models, Mouse, β-galactosidase