Elamipretide (SS-31) Improves Functional Connectivity in Hippocampus and Other Related Regions Following Prolonged Neuroinflammation Induced by Lipopolysaccharide in Aged Rats

Abstract

Neuroinflammation has been recognized as a major cause for neurocognitive diseases. Although the hippocampus has been considered an important region for cognitive dysfunction, the influence of hippocampal neuroinflammation on brain functional connectivity (FC) has been rarely studied. In this study, lipopolysaccharide (LPS) was used to induce systemic inflammation and neuroinflammation in the aged rat brain, while elamipretide (SS-31) was used for treatment. Systemic and hippocampal inflammation were determined using ELISA, while astrocyte responses during hippocampal neuroinflammation were determined by interleukin 1 beta (IL-1β)/tumor necrosis factor alpha (TNFα) double staining immunofluorescence. Oxidative stress was determined by reactive oxidative species (ROS), electron transport chain (ETC) complex, and superoxide dismutase (SOD). Short- (<7 days) and long-term (>30 days) learning and spatial working memory were tested by the Morris water maze (MWM). Resting-state functional magnetic resonance imaging (rs-fMRI) was used to analyze the brain FC by placing seed voxels on the left and right hippocampus. Compared with the vehicle group, rats with the LPS exposure showed an impaired MWM performance, higher oxidative stress, higher levels of inflammatory cytokines, and astrocyte activation in the hippocampus. The neuroimaging examination showed decreased FC on the right orbital cortex, right olfactory bulb, and left hippocampus on day 3, 7, and 31, respectively, after treatment. In contrast, rats with SS-31 treatment showed lower levels of inflammatory cytokines, less astrocyte activation in the hippocampus, and improved MWM performance. Neuroimaging examination showed increased FC on the left-parietal association cortex (L-PAC), left sensory cortex, and left motor cortex on day 7 with the right flocculonodular lobe on day 31 as compared with those without SS-31 treatment. Our study demonstrated that inhibiting neuroinflammation in the hippocampus not only reduces inflammatory responses in the hippocampus but also improves the brain FC in regions related to the hippocampus. Furthermore, early anti-inflammatory treatment with SS-31 has a long-lasting effect on reducing the impact of LPS-induced neuroinflammation.

Keywords: cognition; elamipretide; functional connectivity; lipopolysaccharide; neuroinflammation.

Figure 1

Elamipretide (SS-31) improved cognitive dysfunction induced by lipopolysaccharide (LPS) in the aged rat brain. (A) Detailed processing line. (B) Morris water maze (MWM) training protocol. Spatial acquisition trials and probe trials were performed on both short term (day 1–7) and long term (day 31–37). (C) Rats treated with LPS showed a significant prolonged escape latency compared with those in vehicle (V) group and SS-31 (S) group from day 2 to 5, which was improved by SS-31 treatment from day 4 in LPS + SS-31 (L + S) group. (D,E) Rats treated with LPS showed reduced time spent in the target quadrant and fewer crossovers to this quadrant compared with V group. The reduction in crossovers was improved by SS-31 treatment. (F) In the long term, a significant difference in escape latency was observed on day 35 with rats treated with LPS seemed to have prolonged escape latency. (G,H) Rats treated with LPS spent significantly less time in the target quadrant compared to rats in the V and S groups, as well as showing significantly fewer crossovers than all other groups. Data are presented as mean ± SEMs, and escape latency was analyzed by two-way ANOVA for repeated measurements with the Bonferroni post-hoc analysis. Time spent in the target quadrant and crossovers were analyzed by one-way ANOVA followed with the Bonferroni post-hoc analysis. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 as indicated or vs. V; *p < 0.05, **p < 0.01, ***p < 0.001 vs. L group.

Figure 2

Effects of lipopolysaccharide (LPS) exposure and elamipretide (SS-31) treatment on oxidative stress. (A) LPS exposure caused a significant mitochondrial ROS (mtROS) increase while rats with SS-31 treatment have lower level. (B,C) LPS exposure and SS-31 treatment caused superoxide dismutase (SOD) changes in the hippocampus. (D–F) LPS exposure and SS-31 treatment caused electron transport chain (ETC) complex I/III/IV changes in the hippocampus. Data are presented as mean ± SEMs (n = 5). Comparisons were made by two-way ANOVA followed by the Bonferroni post-hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS (L) group.

Figure 3

Effects of lipopolysaccharide (LPS) exposure and elamipretide (SS-31) treatment. ELISA showed differences in (A,D) the systemic and (B,E) the hippocampus IL-1β and TNFα expression. RT-PCR showed differences in mRNA expression for (C) IL-1β and (F) TNFα Data are presented as mean ± SEMs (n = 5). Comparisons were made by two-way ANOVA followed by the Bonferroni post-hoc analysis. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 as indicated or vs. V; *p < 0.05, **p < 0.01, ***p < 0.001 vs. L group.

Figure 4

Astrocytes activation induced by lipopolysaccharide (LPS) exposure and elamipretide (SS-31) treatment. (A,B) LPS exposure induced a significant increase in IL-1β/TNFα secretion and astrocyte activation both in the LPS (L) and LPS + SS-31 (L + S) groups. SS-31 treatment improved the inflammatory response in the aged rat brain. (C,D) SS-31 treatment induced no significant inflammatory responses and astrocyte activation. (E,F) Double-stained immunofluorescence cell count showed that SS-31 treatment reduced the inflammatory response from day 3 to 30, whereas LPS caused prolonged IL-1β/TNFα secretion and astrocyte activation. (G,H) SS-31 treatment induced no significant change in IL-1β/TNFα positive astrocytes. Data are presented as mean ± SEMs (n = 5). Comparisons were made by two-way ANOVA followed by the Bonferroni post-hoc analysis. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 as indicated or vs. V; *p < 0.05, **p < 0.01, ***p < 0.001 vs. L group.

Figure 5

Lipopolysaccharide (LPS) exposure induced both short- and long-term functional connectivity (FC) changes in the aged rat brain. LPS exposure caused FC changes in (A) 3 days, (B) 7 days, and (C) 31 days. (D–F) Localization for different regions of interest (ROIs) on day 3, 7, and 31. Data were analyzed by one-way ANOVA followed by the post-hoc two-sample t-test. A voxel-level height threshold of p < 0.001 and a cluster-extent threshold of 20 were considered as statistically significant. Both the left and right hippocampus were used as seed voxels.

Figure 6

SS-31 treatment induced both short- and long-term functional connectivity (FC) changes in the aged rat brain. (A) SS-31 treatment caused FC changes in 3 days. SS-31 treatment improved FC connectivity in (B) 7 days and (C) 31 days. (D–F) Localization for different regions of interest (ROIs) on day 3, 7, and 31. Data were analyzed by one-way ANOVA followed by the post-hoc two-sample t-test. A voxel-level height threshold of p < 0.001 and a cluster-extent threshold of 20 were considered as statistically significant. Both the left and right hippocampus were used as seed voxels.

Figure 7

Relationship between functional connectivity (FC) and behavior performances on day 7. (A) Correlation analysis in L and NS groups. (B) Correlation analysis in L and L + S groups. Pearson's correlation was used with p < 0.05 for statistical significance and r² for correlation.

Figure 8

Relationship between functional connectivity (FC) and behavior performances on day 31. (A) Correlation analysis in L and NS groups. (B) Correlation analysis in L and L + S groups. Pearson's correlation was used with p < 0.05 for statistical significance and r² for correlation.