Microglia mediate postoperative hippocampal inflammation and cognitive decline in mice

Abstract

Surgery can induce cognitive decline, a risk that increases with advancing age. In rodents, postoperative cognitive decline (POCD) is associated with the inflammatory activation of hippocampal microglia. To examine the role of microglia in POCD, we inhibited the colony-stimulating factor 1 receptor (CSF1R) in adult mice, effectively depleting CNS microglia. Surgical trauma (tibial fracture) reduced the ability of mice to remember a conditioned response learned preoperatively, a deficit more pronounced and persistent in mice with diet-induced obesity (DIO). Whereas microglial depletion by itself did not affect learning or memory, perioperative microglial depletion remarkably protected mice, including those with DIO, from POCD. This protection was associated with reduced hippocampal levels of inflammatory mediators, abrogation of hippocampal recruitment of CCR2+ leukocytes, and higher levels of circulating inflammation-resolving factors. Targeting microglia may thus be a viable strategy to mitigate the development of POCD, particularly in those with increased vulnerability.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Perioperative microglial depletion prevents surgically induced memory loss in mice.

(A) Representative hippocampal immunofluorescence images including Iba1+ cells (green) and nuclei (DAPI; blue) from mice following tibial fracture, showing marked depletion of hippocampal microglia in response to PLX5622 treatment for 7 days before surgery and 3 days following surgery. Scale bars: 50 mm. (B) Quantification of the extent of microglial depletion in A (n = 6/group; *P < 0.01 vs. control by two-tailed Student’s t test). (C) Schematic, depicting the protocol for preoperative trace-fear conditioning (TFC) training, TFC testing, experimental surgery, and postoperative analyses of both hippocampal and systemic inflammation and the memory of preoperative associative learning. (D and E) Freezing in response to contextual cues, expressed as a percentage of total time in the testing environment prior to surgery (D) and relative to that of appropriate sham-treated controls3 days following surgery (E). Data in D were analyzed by two-way ANOVA. Data in E were analyzed by two-tailed Students’ t test (***P = 0.0002 for surgery vs. sham-treated control). For both D and E,n = 10/group.

Figure 2. Perioperative microglial depletion abrogates surgically induced hippocampal inflammation.

(A) Tissue ELISA, showing the rise in hippocampal levels of both IL-6 and MCP-1 in response to surgery and the prevention of this postoperative rise by perioperative PLX5622 treatment as analyzed by two-way ANOVA (P = 0.006 at 24 hours for IL-6, and P < 0.01 at both 6 and 24 hours for MCP- 1). (B) Plasma ELISA, showing the comparative lack of effect of PLX5622 treatment on postoperative plasma IL-6 and MCP-1 levels. Data were analyzed by two-way ANOVA. In all cases, n = 7–8/group, and ***P < 0001; **P < 0.001 for surgery vs. corresponding sham-treated control.

Figure 3. Perioperative PLX5622 treatment prevents surgery-induced entry of CCR1-expressing cells into the hippocampal region of the brain in mice.

(A) Representative immunofluorescence images of hippocampal sections from CCR2RFP/+: Cx3CR1GFP/+ mice (n = 4, 3 sections per sample), showing that sham-treated mice do not have CCR2-expressing (RFP+) cells in the hippocampus or associated choroid plexus. (B) Similar images in mice 24 hours after surgery, showing that depletion of microglia (green) by perioperative PLX5622 treatment is associated with the complete disappearance of CCR2-expressing cells otherwise present in the choroid plexus and entering the hippocampus at this time point. Scale bars: 50 mm. (C) Quantification of the prevention of surgically induced CCR2RFP/+ cell infiltration by PLX5622 treatment in mice from B (n = 8 mice/group; *P < 0.05 vs. control by two-tailed Student’s t test). (D) FACS data from blood samples taken from control and PLX5622-treated mice 3 days following surgery, showing no effect of perioperative PLX5622 treatment on the number of circulating CCR2RFP/+ cells (n = 8 mice/group; analyzed by two-tailed Student’s t test).

Figure 4. Perioperative microglial depletion prevents the inflammatory and cognitive consequences of surgery in mice with DIO.

(A) The freezing response to preoperative TFC testing (% of total time) of healthy (chow-fed) controls, mice with DIO, and mice with DIO treated with PLX5622 (PLX), showing no difference between groups. (B) Freezing times measured from the mice in A (% vs. corresponding sham-treated mice) during TFC testing 3 and 7 days after surgery. Whereas the impact of surgery on TFC memory is transient and returnsto sham levels by day 7 in control mice (*P < 0.001 vs. control at day 3), mice with DIO have a more pronounced (*P < 0.001 vs. control at day 3) and persistent ($$P < 0.001 vs. control mice at day 7) memory loss. Perioperative PLX5622 treatment protects mice with DIO from surgery induced memory loss (‡‡P < 0.005 vs. DIO alone at day 7). (C and D) Analysis of LXA4 and IL-6 levels, showing that DIO paradoxicallylowers plasma LXA4 levels that otherwise rise by 3 days after surgery in healthy control mice (C) and potentiates the postoperative (day 3) rise in hippocampal IL-6 levels seen in control mice (D). (E and F) Corresponding measurements from plasma (LXA4) and hippocampus (IL-6), showing that perioperative PLX5622 treatment abolishes the impact of surgery on circulating LXA4 levels in mice with DIO (E) and prevents hippocampal IL-6 levels from rising in mice with DIO (F). All data are from n = 12–19/group and were analyzed by two-way ANOVA (A, C, and D), one-way ANOVA (B), and Student’s t test (E and F); **P < 0.001 for comparisons shown.