Effects of aging on motor and exploratory behavior in mice

In the study, the OFT was used to observe the effects of aging on motor activity and exploratory behavior in mice. The results showed that the mean moving distance (m) (Fig. 1A; F(3,41) = 7.698, P < 0.01), the mean moving speed (m/s) (Fig. 1B; F(3,41) = 7.685, P < 0.01), the number of line crossing (Fig. 1C; F(3,41) = 7.035, P < 0.01), and standing up (Fig. 1D, F(3,41) = 15.72, P < 0.01) had significant effects, and they were significantly decreased in 20 M and 24 M mice compared to 6 M mice. In addition, the moving distance and the mean moving speed were significantly decreased, while the number of lines crossing and number of standing were not significantly decreased in 16 M compared to 6 M mice. These data demonstrated an aging phenomenon: 16 M mice showed a mild decrease in motor ability, while the effects were much more pronounced in 20 M and 24 M mice.

Fig. 1

Effects of aging on motor activity and exploratory behavior in 6 M, 16 M, 20 M and 24 M mice (open field test). A The total moving distance (m). B The mean moving speed (m/s). C The number of lines crossing. D The number of standing. Results are expressed as mean ± SD. 6 M, 16 M, 20 M, n = 12; 24 M, n = 9. **P < 0.01 vs 6 M control

Effects of aging on learning and memory abilities in mice

The MWM was used to investigate changes in learning and memory abilities during the aging process in mice. In the orientation navigation experiment, compared with the first day (d1), the escape latency in 6 M, 16 M and 20 M group had a decreasing trend and significantly decreased on d4 in 6 M group (Fig. 2A; F(3,44) = 4.746, P < 0.01), on d3 in 16 M group (Fig. 2A; F(3,44) = 4.556, P < 0.05), and on d4 in 20 M group (Fig. 2A; F(3,44) = 3.885, P < 0.05). However, the escape latency in 24 M group had no decreasing trend from d1 to d4. Additionally, the escape latency had significant effects on d4 (Fig. 2A; F(3,41) = 3.57, P < 0.05). And compared with 6 M group, the escape latency was significantly prolonged in 20 M and 24 M mice on d4 (Fig. 2A; P < 0.05), and had no significant effects on d1-d3. In the space exploration experiment on d5, compared with 6 M mice, the latency of first entry to the platform (LFP, s) was significantly increased in 16 M, 20 M, and 24 M mice (Fig. 2B, C; F(3,41) = 3.617, P < 0.05 or P < 0.01). The swimming time in the quadrant with platform (STP, s) (Fig. 2D; F(3,41) = 4.416, P < 0.05 or P < 0.01) and the number of crossing the platform (NCP) (Fig. 2E; F(3,41) = 3.55, P < 0.05) were significantly decreased in 20 M and 24 M compared with 6 M mice. These results suggested that the learning and memory ability was mild decreased in 16 M mice, but was significantly impaired in 20 M and 24 M mice, especially in the 24 M group.

Fig. 2

Effect of aging on learning and memory impairments in 6 M, 16 M, 20 M and 24 M mice (Morris water maze). A The mean escape latency (s) in the orientation navigation experiment. B Representative path of probe trial experiments on day 5. C The latency of first entry to the platform (s). D The swimming time in the quadrant of platform (s). E The number of crossing the platform. Results are expressed as mean ± SD. 6 M, 16 M, 20 M, n = 12; 24 M, n = 9. *P < 0.05, **P < 0.01 vs 6 M group; #P < 0.05, ##P < 0.01 vs d1 in the orientation navigation experiment

Effects of aging on neuronal degeneration in the cortex and hippocampus in mice

H&E and Nissl staining were performed to examine neuropathological changes in the cortex and hippocampus during aging. Based on H&E staining, there were a few neuronal abnormalities in the cortex and hippocampal CA1 and CA3 regions in 6 M mice. Compared with 6 M mice, there were no obvious increase of pathological damages in 16 M mice. However, there were obvious pathological damages in the cortex and hippocampal CA1 and CA3 regions in 20 M and 24 M mice, especially in the 24 M group. More neurons exhibited nuclear pyknosis and hyperchromatic nuclei in cortex and CA3 regions in 24 M mice, and eosinophilic degeneration in CA3 region in 24 M mice (Additional file 2: Fig. S2).

Nissl staining is a well-known method that specifically stains Nissl bodies and is often used to identify neuronal damage [23]. Nissl staining showed abundant Nissl bodies in the cortex and hippocampal CA1 and CA3 regions in 6 M and 16 M mice. When compared with 6 M mice, the number of Nissl bodies was significantly reduced in cortex (Fig. 3A, B; F(2,9) = 243.9, P < 0.01) and hippocampal CA1 (Fig. 3C; F(2,9) = 99.79, P < 0.05 or P < 0.01) and CA3 (Fig. 3D; F(2,9) = 71.02, P < 0.05 or P < 0.01) regions in 20 M and 24 M mice, especially the 24 M group. These results suggested that there were no significant neuronal damages before 16 M, but obvious neuronal damages were observed at the age of 20 M and 24 M in mice.

Fig. 3

Effects of aging on changes of Nissl bodies in the cortex and hippocampus in mice (Nissl staining, 400 × , scale bar = 20 μm). A The results of Nissl staining in the cortex, hippocampus CA1 and CA3 in 6 M, 16 M, 20 M and 24 M mice. B The mean density of Nissl bodies in the cortex. C The mean density of Nissl bodies in hippocampus CA1. D The mean density of Nissl bodies in hippocampus CA3. Results are expressed as mean ± SD, n = 4. *P < 0.05, **P < 0.01 vs the 6 M control. AU presents an arbitrary unit

Effects of aging on senescence-associated β-gal expression in the cortex and hippocampus in mice

The β-gal is an important biomarker for the senescence of neurons. β-gal activity is significantly increased in aging hippocampal neurons in vitro [24]. Our results showed that β-gal activity was relatively low in the cortex and hippocampal CA1 and CA3 regions in 6 M and 16 M mice. Compared with younger 6 M mice, β-gal activity was significantly increased in the cortex (Fig. 4A, B; F(3,8) = 52.74, P < 0.05 or P < 0.01) and hippocampal CA1 (Fig. 4C; F(3,8) = 22.67, P < 0.05 or P < 0.01) and CA3 (Fig. 4D; F(3,8) = 25.16, P < 0.05 or P < 0.01) regions in 20 M and 24 M mice, especially in the 24 M group. The results suggested that there was no significant neuronal senescence in 16 M mice, but obvious neuronal senescence was appeared at the age of 20 M and 24 M in mice.

Fig. 4

Effects of aging on β-gal activity in the cortex and hippocampus in mice. A The β-gal staining in the cortex, hippocampus CA1 and CA3 in the 6 M, 16 M, 20 M and 24 M mice (400 × , scale bar = 20 μm). B The mean density of β-gal in the cortex. C The mean density of β-gal in hippocampus CA1. D The mean density of β-gal in hippocampus CA3. Results are expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs the 6 M control

Effects of aging on MAP2 expression in the cortex and hippocampus in mice

The MAP2 is an important biomarker located in neuronal dendrites. MAP2 expression in the hippocampus and cortex is significantly decreased in old rats [25]. Therefore, we further detected MAP2 expression in the cortex and hippocampal CA1 and CA3 regions using immunohistochemistry. The results showed that the expression of MAP2 was abundant in the cortex and hippocampal CA1 and CA3 regions in 6 M and 16 M mice (Fig. 5). Compared to 6 M mice, the expressions of MAP2 were significantly reduced in the cortex (Fig. 5A, B; F(2,9) = 14.16, P < 0.05 or P < 0.01) and hippocampal CA1 (Fig. 5C; F(2,9) = 10.67, P < 0.05 or P < 0.01) and CA3 (Fig. 5D; F(2,9) = 6.263, P < 0.05 or P < 0.01) regions in 20 M and 24 M mice, especially the 24 M group. These findings suggested that the expression of MAP2 in neurons might significantly decrease when the mice entered older age.

Fig. 5

Effects of aging on MAP2 expression in the cortex and hippocampus in mice (immunohistochemistry, 400 × , scale bar = 20 μm). A The expression of MAP2 in the cortex, hippocampus CA1 and CA3 in 6 M, 16 M, 20 M and 24 M mice. B The mean density of MAP2 in the cortex. C The mean density of MAP2 in hippocampus CA1. D The mean density of MAP2 in hippocampus CA3. Results are expressed as mean ± SD, n = 4. *P < 0.05, vs the 6 M control

Effects of aging on NLRP1, ASC, caspase-1, and IL-1β expression in the hippocampus in mice

In order to confirm whether NLRP1 inflammasome activation is involved in aging-related neuronal damage, we further investigated the expressions of NLRP1, ASC, caspase-1, and IL-1β in the hippocampus. The results showed that the expression of NLRP1 was significantly increased in 20 M and 24 M mice, especially in the 24 M group, compared with 6 M (Fig. 6A, B; F(3,8) = 8.872, P < 0.05). In addition, the expressions of ASC, caspase-1, and IL-1β gradually increased with aging; they were significantly increased in 20 M and 24 M mice (ASC: Fig. 6A, C; F(3,8) = 5.362, P < 0.05; caspase-1: Fig. 6D; F(3,8) = 7.365, P < 0.05; and IL-1β: Fig. 6E; F(3,8) = 6.496, P < 0.05) compared to 6 M mice. While in 16 M group, these parameters had no significant changes compared with 6 M mice. The data suggested that NLRP1 inflammasome activation was closely involved in aging-related neuronal damage during aging process.

Fig. 6

Effects of aging on the expressions of NLRP1, ASC, caspase-1and IL-1β in the hippocampus in mice. A The bands of NLRP1, ASC, caspase-1, IL-1β and β-actin examined by immunoblot in 6 M, 16 M, 20 M and 24 M mice. B The relative expression of NLRP1 over 6 M. C The relative expression of ASC over 6 M. D The relative expression of caspase-1 over 6 M. E The relative expression of IL-1β over 6 M. Results are expressed as mean ± SD, n = 3. *P < 0.05 vs the 6 M control

Effects of aging on ROS production in the cortex and hippocampus in mice

Given that ROS plays crucial roles in neuroinflammation and neuronal damage, we also measured ROS production in the cortex and hippocampus via DHE fluorescence staining. The results showed that there was little ROS production in the cortex and hippocampal CA1 and CA3 regions in 6 M mice. In 16 M mice, ROS production was slightly increased in the cortex, but it was significantly increased in hippocampal CA1 (Fig. 7B, E; F(3,8) = 31.36, P < 0.05) and CA3 (Fig. 7C, F; F(3,8) = 16.56, P < 0.05) regions. In 20 M and 24 M mice, ROS production was significantly increased more than tenfold in the cortex (Fig. 7A, C; F(3,8) = 22.17, P < 0.01) and hippocampal CA1 (Fig. 7B, E; F(3,8) = 31.36, P < 0.01) and CA3 (Fig. 7C, F; F(3,8) = 16.56, P < 0.01) regions compared with 6 M mice. These results suggested that excessive ROS accumulation was closely involved in neuronal damage during aging.

Fig. 7

Effects of aging on ROS production in the cortex and hippocampus in mice (DHE staining, 400 × , scale bar = 50 μm). A–C The ROS production in the cortex, hippocampus CA1 and CA3 in 6 M, 16 M, 20 M and 24 M mice. D The mean density of ROS production in the cortex. E The mean density of ROS production in hippocampus CA1. F The mean density of ROS production in hippocampus CA3. Results are expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs the 6 M control

Effects of aging on NOX2, p22phox, and p47phox expression in the hippocampus in mice

The NOX2 is a key enzyme in the process of ROS generation in the brain. Hence, we further measured the effect of senescence on the expressions of NOX2, p22phox, and p47phox proteins in the hippocampus via western blotting. The results showed that the expressions of NOX2, p22phox, and p47phox were relatively low in 6 M and in 16 M mice. Compared with 6 M mice, the expressions of NOX2 (Fig. 8A, B; F(3,8) = 5.791, P < 0.05), p22phox (Fig. 8C; F(3,8) = 10.06, P < 0.05), and p47phox (Fig. 8D; F(3,8) = 14.75, P < 0.05 or P < 0.01) were significantly increased in 20 M and 24 M mice. The change of NOX2 was consistent with the ROS production in the brain during aging. These results suggested that NOX2 was closely involved in ROS generation and accumulation in brain during aging.

Fig. 8

Effects of aging on the expressions of NOX2, p22phox and p47phox in the hippocampus in mice (immunoblot). A The bands of NOX2, p22phox, p47phox and β-actin in 6 M, 16 M, 20 M and 24 M mice. B The relative expression of NOX2 over 6 M. C The relative expression of p22phox over 6 M. D The relative expression of p47phox over 6 M. Results are expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs the 6 M control