Maresin 1 attenuates neuroinflammation in a mouse model of perioperative neurocognitive disorders

Abstract

Background: Resolution of inflammation is an active and dynamic process after surgery. Maresin 1 (MaR1) is one of a growing number of specialised pro-resolving lipids biosynthesised by macrophages that regulates acute inflammation. We investigated the effects of MaR1 on postoperative neuroinflammation, macrophage activity, and cognitive function in mice.

Methods: Adult male C57BL/6 (n=111) and Ccr2RFP/+Cx3cr1GFP/+ (n=54) mice were treated with MaR1 before undergoing anaesthesia and orthopaedic surgery. Systemic inflammatory changes, bone healing, neuroinflammation, and cognition were assessed at different time points. MaR1 protective effects were also evaluated using bone marrow derived macrophage cultures.

Results: MaR1 exerted potent systemic anti-inflammatory effects without impairing fracture healing. Prophylaxis with MaR1 prevented surgery-induced glial activation and opening of the blood-brain barrier. In Ccr2RFP/+Cx3cr1GFP/+ mice, fewer infiltrating macrophages were detected in the hippocampus after surgery with MaR1 prophylaxis, which resulted in improved memory function. MaR1 treatment also reduced expression of pro-inflammatory cell surface markers and cytokines by in vitro cultured macrophages. MaR1 was detectable in the cerebrospinal fluid of older adults before and after surgery.

Conclusions: MaR1 exerts distinct anti-inflammatory and pro-resolving effects through regulation of macrophage infiltration, NF-κB signalling, and cytokine release after surgery. Future studies on the use of pro-resolving lipid mediators may inform novel approaches to treat neuroinflammation and postoperative neurocognitive disorders.

Keywords: glia; macrophages; neurocognitive disorders; neuroinflammation; omega 3 fatty acids; postoperative complications; pro-resolving lipid mediators.

Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Figures

Fig 1

Anti-inflammatory and pro-resolving effects of maresin 1 prophylaxis. (a) Study design and endpoints. (b–e) Time course of plasma levels of interleukin (IL)-6, IL-12, CXCL1, and IL-10 at 24 h, 72 h, and 14 days after orthopaedic surgery. (f) Cognitive evaluation of mice using contextual fear conditioning. Training was performed before surgery and hippocampus-dependent memory was assessed at 72 h. MaR1 prevented surgery-induced cognitive dysfunction in both wild-type mice and (g) Ccr2RFP/+Cx3cr1GFP/+ transgenic mice. (h–i) Effects of MaR1 on bone healing 14 days after stabilised tibia fracture surgery. Arrows indicate the original cortical bone. Scale bar: 50 μm. Data expressed as mean (sem); *P<0.05; n=5–6 per group (**P<0.01, n=8–10 for behaviour). C, Control; MaR1, Maresin 1; S, Surgery; sem, standard error of the mean.

Fig 2

Maresin 1 prevents hippocampal neuroinflammation by regulating macrophage infiltration and claudin-5 (cld-5) expression at the blood–brain barrier. (a) Representative confocal images immunostained for GFAP and Iba1 at 24 h and 72 h. (b, c) Quantification of GFAP and Iba1 immunostaining. Surgery affected astrocytic processes and microglia/macrophage activation peaking at 24 h, with mild changes at 72 h. MaR1 pretreatment significantly reduced surgery-induced neuroinflammation. (d) Representative z-stack images from Ccr2RFP/+Cx3cr1GFP/+ at 24 h show macrophage infiltration (red) and residency microglia (green) in the CA1–CA3 hippocampus region. (e) Treatment with MaR1 reduced macrophage infiltration into hippocampus at 24 and 72 h after surgery. (f, g) Representative images for cld-5 immunostaining. MaR1 prevented cld-5 downregulation on the hippocampal vasculature, restoring the expression to control levels. Scale bar: 50 μm. Data expressed as mean (sem). *P<0.05, **P<0.01; n=3–5 per group. C, Control; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MaR1, Maresin 1; slm, stratum lacunosum-moleculare; pyr, pyramidale; sr, stratum radiatum; S, Surgery; sem, standard error of the mean.

Fig 3

Maresin 1 effects on bone marrow-derived macrophages (BMDMs). (a) Representative images of immunostaining of NF-κB in BMDMs. Stimulated with 10 ng ml−1 LPS for 2 h. (b) 2 h of LPS stimulation (10 ng ml−1) significantly increased NF-κB nuclear translocation in BMDMs. (c) LPS caused significant increase of TNF-α release from BMDMs after 24 h stimulation, which was reduced by co-application of MaR1 (10 nM). (d) NADPH oxidase mediated superoxide production in BMDMs was significantly increased after 24 h of LPS stimulation, which was inhibited by MaR1 co-application. (e–h) MaR1 reduced LPS-induced PD-L1 and CD86 expression in BMDMs at 24 h. No significant effect of MaR1 on PD-L2 and CD206 expression in BMDMs stimulated by M2 polarisation cytokines (IL-4/IL-10/TGF-β). Data are expressed as mean (sem). *P<0.05, **P<0.01; n=3–5 per group. CLU, chemiluminescence unit; IL, interleukin; LPS, lipopolysaccharide; MaR1, Maresin 1; MFI, median fluorescence intensity; NADPH, nicotinamide adenine dinucleotide phosphate; sem, standard error of the mean; TNF, tumour necrosis factor.

Fig 4

Maresin 1 levels in CSF before, 24 h, and 6 weeks after major non-cardiac, non-neurologic surgery in older patients (age ≥60 yr). (a) Each line represents a single patient. Diagonal cross marks on the X and Y axes indicate non-linearity/scale discontinuity. n=11. (b) Working model for MaR1 protection in PNDs. Pre-treatment with MaR1 regulated excessive inflammation from circulating macrophage by reducing NF-κB activation, oxidative stress, pro-inflammatory cytokine release, overall contributing to a ‘M2-like’ switch. This systemic milieu did not impair fracture healing or cause signs of immunosuppression. In fact, these systemic effects prevented loss of cld-5 expression at the blood–brain barrier (BBB) and macrophage infiltration into the brain parenchyma. Overall, dampening the postoperative neuroinflammation leads to improved cognitive function. CSF, cerebrospinal fluid; IL, interleukin; MaR1, Maresin 1; NF-κB, nuclear factor-kappa B; PNDs, perioperative neurocognitive disorders; ROS, reactive oxygen species.

Supplementary Fig S1

Effects of MaR1 on hippocampal gene expression. (A) Filtering data using a statistical significance threshold of P

Supplementary Fig S2

Postoperative changes in cognition and CSF MaR1 levels. Scatterplot representing on the Y axis the change in each patient's overall continuous cognitive index score from within 1 month before to 6 weeks after surgery. A positive score represents postoperative cognitive improvement and a negative score represents a decrement in postoperative cognition. Colours represent the same individual patient as reported in Fig. 4a in the main article.