3.1 There were no significant changes in the body weights of mice during fluoxetine intervention
No group exhibited a significant change in body weight from before to after the intervention period, and there were no significant differences in body weight between any two groups compared, such as the Tg-NS and Tg+NS groups (p = 0.866), Tg-NS and Tg-FLX groups (p = 0.649), and Tg+NS and Tg+FLX groups (p = 0.755; Fig. 2A).
3.2 There were no significant changes in the exploratory behavior or general activity of mice in the new environment during fluoxetine intervention
There were no significant differences in the central area activity time ratio or the central area distance time ratio between any two groups compared, such as the Tg-NS and Tg+NS groups (p = 0.764 & p = 0.689), Tg-NS and Tg-FLX groups (p = 0.309 & p = 0.68) , and Tg+NS and Tg+FLX groups (p = 0.808 & p = 0.448; Fig. 2B & C).
3.3 Fluoxetine delayed deficiencies in spatial learning and memory ability during early AD
Traces of the location of the mice in the hidden platform test and the probe trial test for the four groups are presented in Fig. 2D. There were no significant differences in the swimming speed between any two groups compared, such as the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.854, p = 0.757 & p = 0.567, respectively; Fig. 2E). The escape latency of the Tg+NS group in the hidden platform test was significantly longer than that in the Tg-NS group (p = 0.001; Fig. 2F), whereas there was no significant difference in escape latency between the Tg-NS group and Tg-FLX group (p = 0.954; Fig. 2F). The escape latency of the Tg+FLX group in the hidden platform test was significantly shorter than that of the Tg+NS group (p = 0.024; Fig. 2F). There was no significant difference in the swimming distance between the Tg-NS group and Tg+NS group in the hidden platform test (p = 0.102; Fig. 2G); however, the swimming distance of the Tg+FLX group was significantly shorter than that of the Tg+NS group in the hidden platform test (p = 0.027; Fig. 2G). The target zone frequency of the Tg+NS group was significantly lower than that of the Tg-NS group (p = 0.013; Fig. 2H), but there was no significant difference in target zone frequency between the Tg-NS group and Tg-FLX group (p = 0.266; Fig. 2H). Importantly, the target zone frequency of the Tg+NS group was significantly lower than that of the Tg+FLX group (p = 0.031; Fig. 2H). There was no significant difference in the swimming time in the target zone between any two groups, including the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.173, p = 0.230 & p = 0.267, respectively; Fig. 2I). There was no significant difference in the quadrant percentage time between any two groups, including the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.149, p = 0.095 & p = 0.596, respectively; Fig. 2J).
3.4 There were no significant changes in mouse brain mass during the fluoxetine intervention
No two groups compared showed a significant difference in brain mass, including the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.586, p = 0.35 & p = 0.697, respectively; Fig. 2K).
3.5 Fluoxetine rescued the decrease in mature oligodendrocytes and reversed the abnormal increase in oligodendrocyte lineage cells in the hippocampus of AD mice
3.5.1 The protein levels of CNPase and MBP in the hippocampus of AD mice
The hippocampal protein levels of CNPase and MBP (17 kDa & 21 kDa) in the Tg+NS group were significantly lower than those in the Tg-NS group (p = 0.008, p = 0.008 & p = 0.003; Fig. 3A & B). There was no significant difference in the protein levels of CNPase or MBP (17 kDa & 21 kDa) in the hippocampus between the Tg-NS group and Tg-FLX group (p = 0.839, p = 0.521 & p = 0.509; Fig. 3A & B). The hippocampal protein levels of CNPase and MBP (17 kDa & 21 kDa) in the Tg+FLX group were significantly higher than those in the Tg+NS group (p = 0.019, p = 0.015 & p = 0.002; Fig. 3A & B).
3.5.2 The total number of oligodendrocyte lineage cells (Olig2+ cells) and mature oligodendrocytes (CNPase+ cells) in the hippocampus
The Olig2+ cells and CNPase+ cells in the hippocampal subregions (CA1, CA2-3 and DG) of the four groups of mice are shown in Fig. 3C & 3D, respectively.
The total numbers of Olig2+ cells and CNPase+ cells in each group are presented in Table 1. In the current study, the observed variance of the individual estimate (OCE2) was less than half of the observed interindividual variance (OCV2), indicating that the sampling was considered optimal.
The total numbers of Olig2+ cells in the CA1 field, CA2-3 fields and DG of the Tg+NS mice were significantly greater than those in the respective areas of the Tg-NS mice (p = 0.037, p = 0.002 & p < 0.001; Fig. 3E). There were no significant differences in the total numbers of Olig2+ cells in the CA1 field, CA2-3 fields or DG between the Tg-NS mice and Tg-FLX mice (p = 0.123, p = 0.844 & p = 0.320; Fig. 3E). There were no significant differences in the total numbers of Olig2+ cells in the CA1 field between the Tg+NS mice and Tg+FLX mice; however, the total numbers of Olig2+ cells in the CA2-3 fields and DG of the Tg+FLX mice were significantly lower than those in the same areas of the Tg+NS mice (p = 0.201, p = 0.021 & p = 0.003; Fig. 3E).
The total numbers of CNPase+ cells in the CA1 field, CA2-3 fields and DG of the Tg+NS mice were significantly lower than those in the respective areas of the Tg-NS mice (p = 0.003, p = 0.012 & p < 0.001; Fig. 3F). There were no significant differences in the total numbers of CNPase+ cells in the CA1 field, CA2-3 fields or DG between the Tg-NS mice and Tg-FLX mice (p = 0.374, p = 0.386 & p = 0.800; Fig. 3F). The total numbers of CNPase+ cells in the CA1 field, CA2-3 fields and DG of the Tg+FLX mice were significantly greater than those in the respective areas of the Tg+NS mice (p = 0.001, p = 0.005 & p < 0.001; Fig. 3F).
3.5.3 The densities of mature Olig2+ and immature Olig2+ cells in the hippocampus
The Olig2+/CNPase+ (mature Olig2+) and Olig2+/CNPase- (immature Olig2+) cells in the hippocampus (CA1, CA2-3 and DG) of the four groups are shown in Fig. 3G.
The densities of Olig2+/CNPase+ cells in the CA1 field, CA2-3 fields and DG of the Tg+NS mice were significantly lower than those in the same areas of the Tg-NS mice (p < 0.001, p = 0.001 & p = 0.012; Fig. 3H). The density of Olig2+/CNPase+ cells in the CA1 field of the Tg-FLX mice was significantly lower than that in the CA1 field of the Tg-NS mice; however, there were no significant differences in the densities of Olig2+/CNPase+ cells in the CA2-3 fields or DG between the Tg-NS mice and Tg-FLX mice (p = 0.015, p = 0.229 & p = 0.869; Fig. 3H). The densities of Olig2+/CNPase+ cells respectively in the CA1 field, CA2-3 fields and DG of the Tg+FLX mice were significantly great than those in the same regions of the Tg+NS mice (p = 0.007, p = 0.011 & p = 0.014; Fig. 3H).
The densities of Olig2+/CNPase- cells in the CA1 field, CA2-3 fields and DG of the Tg+NS mice were significantly greater than those in the same regions of the Tg-NS mice (p = 0.001, p < 0.001 & p < 0.001; Fig. 3I). There were no significant differences in the densities of Olig2+/CNPase- cells in the CA1 field, CA2-3 fields or DG between the Tg-NS mice and Tg-FLX mice (p = 0.147, p = 0.594 & p = 0.863; Fig. 3I). The densities of Olig2+/CNPase- cells in the CA1 field, CA2-3 fields and DG of the Tg+FLX mice were significantly lower than those of the Tg+NS mice (p = 0.009, p < 0.001 & p = 0.001; Fig. 3I).
The ratios of CNPase+ cells to Olig2+ cells in the CA1 field, CA2-3 fields and DG of the Tg+NS mice were significantly lower than those of the Tg-NS mice (p < 0.001, p < 0.001 & p < 0.001; Fig. 3J). The ratio of CNPase+ cells to Olig2+ cells in the CA2-3 fields of the Tg-FLX mice was significantly lower than that in the same region of the Tg-NS mice; however, there were no significant differences in the ratios of CNPase+ cells to Olig2+ cells in the CA1 field or DG between the Tg-NS mice and Tg-FLX mice (p = 0.01, p = 0.362 & p = 0.066; Fig. 3J). The ratios of CNPase+ cells to Olig2+ cells in the CA1 field, CA2-3 fields and DG of the Tg+FLX mice were significantly greater than those of the Tg+NS mice (p < 0.001, p = 0.002 & p < 0.001; Fig. 3J).
3.6 Fluoxetine not only increased SOX10 protein expression in mature oligodendrocytes but also promoted the maturation of newborn oligodendrocytes in the hippocampus of AD mice
3.6.1 The protein levels of NG2 and SOX10 in the hippocampus
There were no significant differences in the hippocampal protein levels of NG2 or SOX10 between any two groups compared, including between the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.975, p = 0.447, p = 0.706, p = 0.679, p = 0.431 & p = 0.839, respectively; Fig. 4A & B).
3.6.2 The protein level of SOX10 in hippocampal oligodendrocytes
SOX10+ cells in the hippocampus (CA1, CA2-3 and DG) of the four groups are shown in Fig. 4C. There were no significant differences in the density of SOX10+ cells in the CA1 field between any two groups compared, including between the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.24, p = 0.764 & p = 0.550; Fig. 4D), and there were no significant differences in the density of SOX10+ cells in the CA2-3 fields or DG between the Tg-NS group and Tg-FLX group (p = 0.861 & p = 0.888; Fig. 4D). However, the densities of SOX10+ cells in the CA2-3 fields and DG of the Tg+NS mice were significantly lower than those of the Tg-NS mice (p = 0.012 & p = 0.016; Fig. 4D), and the densities of SOX10+ cells in the CA2-3 fields and DG of the Tg+FLX mice were significantly higher than those of the Tg+NS mice (p = 0.004 & p = 0.009; Fig. 4D).
The SOX10+/PDGFα+ cells in the hippocampus (CA1, CA2-3 and DG) of the four groups are shown in Fig. 4E. There were no significant differences in the density of SOX10+/PDGFα+ cells in the CA1 field or CA2-3 fields between any two groups compared, including between the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.791, p = 0.43, p = 0.43, p = 0.217, p = 0.53 & p = 0.277; Fig. 4F). The SOX10+/PDGFα+ cell density in the DG of the Tg+NS mice was significantly lower than that in the DG of the Tg-NS mice; however, there were no significant differences in SOX10+ cell density in the CA2-3 fields or DG between the Tg-NS and Tg-FLX groups or between the Tg+NS and Tg+FLX groups (p = 0.028, p = 0.865 & p = 0.055; Fig. 4F).
The SOX10+/CNPase+ cells in the hippocampus (CA1, CA2-3 and DG) of the four groups are shown in Fig. 4G. There were no significant differences in SOX10+/CNPase+ cell density in the CA1 field or DG between any two groups compared, including between the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.756, p = 0.357, p = 0.756, p = 0.194, p = 0.882 & p = 0.245; Fig. 4H). There were no significant differences in SOX10+/CNPase+ cell density in the CA2-3 fields between the Tg-NS mice and Tg-FLX mice (p = 0.400; Fig. 4H). However, the density of SOX10+/CNPase+ cells in the CA2-3 fields of the Tg+NS mice was significantly lower than that in the CA2-3 fields of the Tg-NS mice, and the density of SOX10+/CNPase+ cells in the CA2-3 fields of the Tg+FLX mice was significantly greater than that in the CA2-3 fields of the Tg+NS mice (p = 0.028 & p = 0.039; Fig. 4H).
3.6.3 Newborn oligodendrocytes in the hippocampus
The BrdU+/Olig2+/CNPase+ (newborn mature Olig2+) cells and BrdU+/Olig2+/CNPase- (newborn immature Olig2+) cells in the hippocampus (CA1, CA2-3 and DG) of the four groups of mice are shown in Fig. 4I.
There were no significant differences in BrdU+/Olig2+/CNPase+ cell density in the CA1 field between any two groups compared, including between the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.751, p = 0.307 & p = 0.165; Fig. 4J). The density of BrdU+/Olig2+/CNPase+ cells in the CA2-3 fields of the Tg-FLX mice was significantly greater than that in the Tg-NS mice; however, there were no significant differences in BrdU+/Olig2+/CNPase+ cell density in the CA2-3 fields between the Tg-NS and Tg+NS groups or between the Tg+NS and Tg+FLX groups (p = 0.04, p = 0.086 & p = 0.111; Fig. 4J). Furthermore, there were no significant differences in BrdU+/Olig2+/CNPase+ cell density in the DG between the Tg-NS mice and Tg-FLX mice (p = 0.132; Fig. 4J). However, the density of BrdU+/Olig2+/CNPase+ cells in the DG of the Tg+NS mice was significantly lower than that in the DG of the Tg-NS mice, and the density of BrdU+/Olig2+/CNPase+ cells in the DG of the Tg+FLX mice was significantly higher than that in the DG of Tg+NS mice (p < 0.001 & p = 0.015; Fig. 4J).
There were no significant differences in BrdU+/Olig2+/CNPase- cell density in the CA1 field between any two groups compared, including between the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.553, p = 0.473 & p = 0.732; Fig. 4K). There were no significant differences in BrdU+/Olig2+/CNPase+ cell density in the CA2-3 fields or DG between the Tg-NS and Tg-FLX groups or between the Tg-NS and Tg+NS groups (p = 0.081, p = 0.383, p = 0.055 & p = 0.579; Fig. 4K). However, the densities of BrdU+/Olig2+/CNPase+ cells in the CA2-3 fields and DG of the Tg+FLX mice were significantly lower than those in the same regions of the Tg+NS mice (p = 0.021 & p = 0.008; Fig. 4K).
3.7 Fluoxetine reduced the levels of soluble Aβ40 and Aβ42, the expression of β-amyloid plaques and the ratio of oligodendrocytes expressing p16 in the hippocampus of AD mice
3.7.1 Aβ40, Aβ42 and amyloid plaques in the hippocampus
The levels of Aβ40 and Aβ42 in the hippocampus of the Tg+NS mice were significantly higher than those in the hippocampus of the Tg-NS mice (p < 0.001 & p < 0.001; Fig. 5A). However, there were no significant differences in Aβ40 or Aβ42 levels in the hippocampus between the Tg-NS mice and Tg-FLX mice (p = 0.991 & p = 0.387; Fig. 5A). The levels of Aβ40 and Aβ42 in the hippocampus of the Tg+NS mice were significantly lower than those in the hippocampus of the Tg+FLX mice (p = 0.006 & p = 0.018; Fig. 5A).
As shown in Fig. 5B, no amyloid plaques were found in the hippocampus of the Tg-NS and Tg-FLX mice. In contrast, many large amyloid plaques were observed in the hippocampus of the Tg+NS and Tg+FLX mice, and there were fewer amyloid plaques in the hippocampus of the Tg+FLX mice than in those of the Tg+NS mice (Fig. 5B). In addition, larger amyloid plaques were found in the hippocampus of the Tg+NS mice than in those of the Tg+FLX mice (Fig. 5B).
3.7.2 The protein level of p16 in hippocampal oligodendrocytes
The p16+/Olig2+ cells in the hippocampus (CA1, CA2-3 and DG) of the four groups are shown in Fig. 5C. The p16+/Olig2+ cell ratios in the CA1 field, CA2-3 fields and DG of the Tg+NS mice were significantly greater than those in the same hippocampal regions of the Tg-NS mice (p = 0.004, p = 0.003 & p = 0.01; Fig. 5D). There were no significant differences in p16+/Olig2+ cell ratios in the CA1 field, CA2-3 fields or DG between the Tg-NS and Tg-FLX groups (p = 0.655, p = 0.058 & p = 0.43; Fig. 5D). Furthermore, there was no significant difference in p16+/Olig2+ cell ratio in the CA1 field between the Tg+NS mice and Tg+FLX mice; however, the p16+/Olig2+ cell ratios in the CA2-3 fields and DG of the Tg+FLX mice were significantly lower than those in the same regions of the Tg+NS mice (p = 0.152, p = 0.018 & p = 0.024; Fig. 5D).
3.8 Fluoxetine inhibited the protein expression and activity of GSK3β and the protein expression of LINGO1 and its ligands MAG and MOG in the hippocampus of AD mice but did not affect 5-HT1AR protein expression
3.8.1 The protein levels of GSK3β in the hippocampus
The expression of hippocampal GSK3β protein in the Tg+NS group was significantly greater than that in the Tg-NS group (p < 0.001; Fig. 5E & F). However, there was no significant difference in hippocampal GSK3β protein expression between the Tg-NS group and Tg-FLX group (p = 0.591; Fig. 5E & F). Finally, hippocampal GSK3β protein expression in the Tg+FLX group was significantly lower than that in the Tg+NS group (p < 0.001; Fig. 5E & F).
3.8.2 The protein levels of p-S9-GSK3β in the hippocampus
The expression of hippocampal p-S9-GSK3β protein in the Tg+NS group was significantly lower than that in the Tg-NS group (p = 0.004; Fig. 5E & F), but there was no significant difference in hippocampal p-S9-GSK3β protein expression between the Tg-NS group and Tg-FLX group (p = 0.641; Fig. 5E & F). Importantly, the Tg+FLX group had significantly higher hippocampal p-S9-GSK3β protein expression than the Tg+NS group mice (p = 0.037; Fig. 5E & F).
3.8.3 The protein levels of 5-HT1AR in the hippocampus
There was no significant difference in 5-HT1AR protein expression in the hippocampus between any two groups compared, including between the Tg-NS and Tg+NS groups, Tg-NS and Tg-FLX groups, and Tg+NS and Tg+FLX groups (p = 0.595, p = 0.489 & p = 0.31; Fig. 5E & F).
3.8.4 The protein levels of LINGO1, MAG and MOG in the hippocampus
LINGO1, MAG and MOG protein levels in the hippocampus of the Tg+NS mice were significantly greater than those in the hippocampus of the Tg-NS mice (p = 0.008, p = 0.035 & p = 0.042; Fig. 5E & F). There were no significant differences in hippocampal LINGO1, MAG or MOG protein expression between the Tg-NS group and Tg-FLX group (p = 0.34, p = 0.463 & p = 0.457; Fig. 5E & F). Finally, hippocampal protein expression of LINGO1, MAG and MOG in the Tg+FLX group was significantly lower than that in the Tg+NS group (p = 0.018, p = 0.003 & p = 0.016; Fig. 5E & F).