At the first part of this study, we evaluated the effect of SIS on some behavioral tasks including Forced swimming test (FST), splash test, and Open-field test (OFT), and Hole-board test (HBT). T-test analysis revealed that SIS could induce depressive-like behavior by increasing immobility time of FST (t = 18.74, df = 58, P < 0.001, Fig. 1A), and also decreasing grooming activity of splash test (t = 18.67, df = 58, P < 0.001, Fig. 1B) in comparison to social condition animals. Also, locomotor activity was assessed by using OFT. Results obtained from t-test showed that distance moved (t = 5.57, df = 58, P < 0.001, Fig. 1C), the number of rearings were increased (t = 8.07, df = 58, P < 0.001, Fig. 1D), and time spent in central zone of OFT was decreased in socially isolated animals in comparison to intact mice (t = 5.58, df = 58, P < 0.001, Fig. 1E). In addition, HBT showed that SIS could decrease the number of head-dips in comparison to normal animals (t = 8.20, df = 58, P < 0.001, Fig. 1D).
In the next step, we counted the population of very small embryonic-like stem cells (VSELs) in bone marrow, peripheral blood, and hippocampus samples in both socially isolated and normal animals. Based previous studies, VSELs were identified by flow cytometry as cells with the appropriate size of 2–10 µm (panels A and B, Fig. 2) and lineage negative, SCA-1 positive, and CD45 negative phenotype (panels C-H, Fig. 2). In this study, we estimated that the mean population of VSELs in bone marrow was 0.062%±0.024 in normal animals and in socially isolated animals the mean population of VSELs in bone marrow was 0.191%±0.06. T-test analysis showed that socially isolated animals had a significantly higher level of VSELs in their bone marrow in comparison to normal mice (t = 10.83, df = 58, P < 0.001, Fig. 2I). In addition, data obtained from peripheral samples showed that the mean population of VSELs was 0.021%±0.014 in normal animals and was 0.034%±0.012 in socially isolated animals. As depicted in Fig. 2J, VSELs counts were significantly higher in socially isolated animals in peripheral blood samples in comparison to normal mice (t = 3.62, df = 58, P < 0.001, Fig. 2J). Also, in hippocampus samples, we observed that the mean population of VSELs was 2.05%±0.18 and 2.46%±0.4 in normal and socially isolated animals, respectively. T-test analysis showed that socially isolated animals had significantly higher levels of VSELs in the hippocampus in comparison to normal mice (t = 5.08, df = 58, P < 0.001, Fig. 2K). In addition, we used correlation study between VSELs counts in bone marrow samples and peripheral blood samples to clarify the mobilization of VSELs between bone marrow and peripheral blood. As illustrated in Fig. 3, the analysis revealed a direct correlation between the population of VSELs in the bone marrow and peripheral blood samples (P < 0.001, r = 0.8384, and 95% confidence interval = 0.6851 to 0.9206). These results strongly suggested the mobilization of VSELs between bone marrow and peripheral blood in SIS.
On the other hand, we evaluated the level of cytokines and nitrite level in both plasma and hippocampus in socially isolated and normal mice. Our results revealed that socially isolated animals had significant higher plasma levels of TNF-α (t = 14.10, df = 58, P < 0.001, Fig. 4A), IL-1β (t = 8.52, df = 58, P < 0.001, Fig. 4C), and IL-6 (t = 9.98, df = 58, P < 0.001, Fig. 4E) as well as nitrite levels (t = 18.74, df = 11.69, P < 0.001, Fig. 4G) in comparison to normal animals. In addition, we have assessed the levels of pro-inflammatory cytokines and nitrite levels in mice hippocampus. T-test analysis revealed that TNF-α (t = 3.04, df = 58, P < 0.01, Fig. 4B), IL-1β (t = 15.54, df = 58, P < 0.001, Fig. 4D), IL-6 (t = 6.64, df = 58, P < 0.001, Fig. 4F), and nitrite levels (t = 16.75, df = 58, P < 0.001, Fig. 4H) were significantly higher in socially isolated animals in comparison to normal animals.
Table 1 shows the correlation analysis between the VSELs population of bone marrow, peripheral blood, as well as hippocampus and other behavioral or molecular assessments in both normal and socially isolated animals. Correlation test showed that data obtained from the behavioral tasks or molecular assessments had not any significant correlation with VSELs population of bone marrow in normal mice (P > 0.05, Table 1). In addition, the same analysis was used to clarify the correlations between behavioral tasks or molecular assessments and VSELs counts in peripheral blood samples. As previous results, the analysis failed to show any significant correlation between VSELs counts of peripheral blood samples and all behavioral tasks or molecular assessments (P > 0.05, Table 1). Also, we evaluated the correlation analysis between VSELs counts of the hippocampus and other factors. As shown in Table 1, correlation analysis failed to show any significant correlation between all behavioral tasks (P > 0.05) and hippocampal VSELs population in normal animals. Moreover, no significant correlation was observed between hippocampal levels of VSELs and TNF-α, IL-1β, IL-6, as well as nitrite (P > 0.05).
In the next step, we evaluated the correlation between VSELs population in the bone marrow and behavioral tasks as well as molecular assessments in socially isolated animals. Correlation test showed that the VSELs counts directly correlated with grooming activity of Splash test (P < 0.01), the number of head dips in Hole-board test (P < 0.001), and time in the central zone of OFT (P < 0.01). Also, we observed that VSELs population in bone marrow samples inversely correlated with immobility time of FST (P < 0.001), distance moved of OFT (P < 0.01) as well as the number of rearing of OFT (P < 0.001). Interestingly, our results revealed that the plasma level of TNF-α significantly correlated with VSELs counts (P < 0.05). In addition to TNF-α, we observed that the plasma levels of IL-1β significantly correlated with VSELs counts in bone marrow samples (P < 0.05 and P < 0.01, respectively). Also, the analysis showed that nitrite levels of plasma samples inversely correlated with VSELs population in bone marrow samples of socially isolated animals (P < 0.05). In this study, we did not observe any significant correlation between plasma levels of IL-6 and VSELs population of bone marrow; however, this analysis suggested a marginally significant correlation (P = 0.056). All results and statistically reports were shown in Table 1.
On the other hand, we evaluated the correlations between VSELs counts of peripheral blood samples and behavioral tasks as well as molecular assessments in socially isolated animals. As depicted in Table 1, VSELs counts of peripheral blood samples had a significant inverse correlation with immobility time of FST (P < 0.05), distance moved of OFT (P < 0.05), and the number of rearing of OFT (P < 0.01). Also, the analysis showed a direct correlation between VSELs counts of peripheral blood samples and grooming activity of Splash test (P < 0.01), number of head dips in Hole-board test (P < 0.001), and time in the central zone of OFT (P < 0.001). In plasma samples, we observed that TNF-α, IL-1β, and nitrite level had a significant inverse correlation with VSELs counts of peripheral blood samples (P < 0.05, P < 0.01, and P < 0.001, respectively). Also, analysis revealed a marginally significant inverse correlation between IL-6 and VSELs counts of peripheral blood samples in socially isolated animals (P = 0.051).
In the final step, the correlation study demonstrated that the hippocampal VSELs levels inversely correlated with immobility time in FST (P < 0.001), distance moved and the number of rearings in OFT (P < 0.05 and P < 0.001, respectively). On the other hand, direct correlations were observed between VSELs population of hippocampus and grooming activity in Splash test (P < 0.05), number of head dips in HBT (P < 0.01), and distance moved in OFT (P < 0.05). In addition, we observed that VSELs inversely correlated with pro-inflammatory cytokines including TNF-α (P < 0.01), IL-1β (P < 0.01), and IL-6 (P < 0.05) as well as nitrite level (P < 0.01) in the hippocampus. All statistical analysis reports were mentioned in Table 1.