Long-term treadmill exercise improves spatial memory and increases SOX2-positive cells in the hippocampus of middle-aged male mice.

Age-related cognitive decline is closely associated with hippocampal deterioration, yet the cellular and molecular adaptations underlying exercise-induced cognitive improvement remain incompletely understood. In the present study, nine-month-old male C57BL/6 J mice were randomly assigned to a treadmill exercise group or a sedentary control group and trained for four months, reaching approximately 13 months of age at outcome assessment. Cognitive functions were assessed using the Morris water maze, and hippocampal tissue was examined by Nissl staining and immunofluorescence for SOX2 and GFAP. Database-based network analysis was performed to identify candidate targets shared between exercise-associated and hippocampus-associated gene sets, and the corresponding mouse genes were assessed by RT-qPCR. As a result, long-term treadmill exercise shortened escape latency, reduced swimming distance on the fifth acquisition day, and increased platform crossings during the probe trial. Exercised mice also exhibited fewer hippocampal neurons with condensed nuclei and more SOX2-positive cells than controls. The molecular network analysis identified 48 overlapping human targets, with TNF, IL6, IL1B, PTGS2, TP53, MMP9, TLR4, HMOX1, IGF1, and NFE2L2 ranked as the top hub targets. RT-qPCR confirmed altered hippocampal expression of eight of the ten corresponding mouse genes. Collectively, these findings link improved spatial memory in long-term treadmill exercise with changes in hippocampal neuronal morphology, SOX2-positive cells, and genes related to inflammation, oxidative stress, and cellular stress responses in middle-aged male mice.