Neuroprotectin D1 Protects Against Postoperative Delirium-Like Behavior in Aged Mice

Abstract

Postoperative delirium (POD) is the most common postoperative complication affecting elderly patients, yet the underlying mechanism is elusive, and effective therapies are lacking. The neuroinflammation hypothesis for the pathogenesis of POD has recently emerged. Accumulating evidence is supporting the role of specialized proresolving lipid mediators (SPMs) in regulating inflammation. Neuroprotectin D1 (NPD1), a novel docosahexaenoic acid (DHA)-derived lipid mediator, has shown potent immunoresolvent and neuroprotective effects in several disease models associated with inflammation. Here, using a mouse model of POD, we investigated the role of NPD1 in postoperative cognitive impairment by assessing systemic inflammatory changes, the permeability of the blood-brain barrier (BBB), neuroinflammation, and behavior in aged mice at different time points. We report that a single dose of NPD1 prophylaxis decreased the expression of tumor necrosis factor alpha TNF-α and interleukin (IL)-6 and upregulated the expression of IL-10 in peripheral blood, the hippocampus, and the prefrontal cortex. Additionally, NPD1 limited the leakage of the BBB by increasing the expression of tight junction (TJ)-associated proteins such as ZO-1, claudin-5, and occludin. NPD1 also abolished the activation of microglia and astrocytes in the hippocampus and prefrontal cortex, which is associated with improved general and memory function after surgery. In addition, NPD1 treatment modulated the inflammatory cytokine expression profile and improved the expression of the M2 marker CD206 in lipopolysaccharide (LPS)-stimulated macrophages, which may partly explain the beneficial effects of NPD1 on inflammation. Collectively, these findings shed light on the proresolving activities of NPD1 in the pro-inflammatory milieu both in vivo and in vitro and may bring a novel therapeutic approach for POD.

Keywords: macrophage polarization; neuroinflammation; neuroprotectin D1; postoperative delirium; specialized proresolving lipid mediators.

Figure 1

Surgery/anesthesia induces postoperative delirium (POD)-like behavior of aged mice, which can be ameliorated by preemptive administration of neuroprotectin D1 (NPD1). At 6, 9, and 24 h after surgery/anesthesia, the buried food test (A), open field test (B–D), and Y maze test (E–G) were executed ordinally. Data are presented as individuals and the lines mark the mean ± standard error of the mean (SEM). Statistics: two-way analysis of variance (ANOVA) followed by Bonferroni post hoc comparison. (A–G) n = 8–10 per group. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group, ^#P < 0.05 vs. the surgery group.

Figure 2

Effects of NPD1 on the expression of inflammatory cytokines in vivo. NPD1 pretreatment alleviated the surgery-induced upregulation of pro-inflammatory factors and promoted the expression of anti-inflammatory factors both in the peripheral blood and the central locations such as the hippocampus and prefrontal cortex at different time points (A–I). The cytokines in peripheral blood and brain tissues were measured by enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± SEM. Statistics: two-way ANOVA followed by Bonferroni post hoc comparison. (A–I) n = 4–5 per group. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group, ^#P < 0.05 vs. the surgery group.

Figure 3

NPD1 protects against the leakage of the blood–brain barrier (BBB) induced by surgery/anesthesia in the hippocampus and prefrontal cortex. Immunostaining of blood vessels (isolectin B4, green) and intravenously injected dextran (10 kDa, red) in brain sections of the hippocampus at 6 h after surgery (A). The arrowhead marked area indicates that the dextran was extravascular. The spectrophotometric quantification of extravascular dextran (10 kDa) levels in the extraction of the hippocampus and prefrontal cortex showed that surgery/anesthesia increased the permeability of the BBB compared with the control, and pretreatment with NPD1 attenuated this phenomenon (B,C). Data are presented as mean ± SEM. Statistics: two-way ANOVA followed by Bonferroni post hoc comparison. (B,C) n = 4–5 per group. *P < 0.05 vs. the control group, ^#P < 0.05 vs. the surgery group. Scale bars represent 50 μm in (A).

Figure 4

NPD1 modulates the expression of tight junction (TJ)-associated proteins in the hippocampus and prefrontal cortex after surgery. Representative Western blotting bands of the expression of occludin, claudin-5, and ZO-1 in the hippocampus and prefrontal cortex at 6 and 9 h after surgery (A,B). Quantification analyses of the expression of occludin, claudin-5, and ZO-1 were normalized to that of β-actin as internal control (C–H). Data are presented as mean ± SEM. Statistics: two-way ANOVA followed by Bonferroni post hoc comparison. (C–H) n = 4–5 per group. **P < 0.01 vs. the control group, ^#P < 0.05 vs. the surgery group, ^##P < 0.01 vs. the surgery group.

Figure 5

NPD1 pretreatment reverses the activation of astrocytes and microglia in the hippocampus and prefrontal cortex elicited by surgery/anesthesia. Representative images of immunofluorescence showed the expression of glial fibrillary acidic protein (GFAP) and Iba-1 in the hippocampus and prefrontal cortex at 24 h after surgery (A). Surgery/anesthesia caused distinct changes in the morphology of glial cell, including shortened processes and a reduced cell soma in astrocytes and an amoeba-like morphology in microglia. Preemptive administration of NPD1 significantly restored the classic stellate shape of astrocytes and the ramified shape of microglia. Quantification results of immunostaining at 24 h after surgery shown in (B–E). Data are presented as mean ± SEM. Statistics: two-way ANOVA followed by Bonferroni post hoc comparison. (B–E) n = 4–5 per group. *P < 0.05 vs. the control group, ^#P < 0.05 vs. the surgery group. Scale bars represent 100 μm in (A).

Figure 6

NPD1 promotes macrophage polarization to the M2 phenotype after pro-inflammatory stimulation of BMDMs and modulates the expression of inflammatory cytokines. NPD1 promoted the transformation of BMDM cell markers from the M1 to M2 phenotypes, as detected by flow cytometry (A,B). The ratios of M1 and M2 phenotypes in different groups were calculated (C,D). The production of tumor necrosis factor-α (TNF-α), interleukin (IL)-12, and IL-10 by BMDMs were tested by ELISA (E–G). Data are presented as mean ± SEM. Statistics: two-way ANOVA followed by Bonferroni post hoc comparison. (A–G) n = 5 per group. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group, ^#P < 0.05 vs. the surgery group, ^##P < 0.01 vs. the surgery group.