Maresin 1 attenuates neuroinflammation in a mouse model of perioperative neurocognitive disorders  T. Yang, G. Xu, P.T. Newton, A.S. Chagin, S. Mkrtchian, M. Carlström, X.-M. Zhang, R.A. Harris, M. Cooter, M. Berger, K.R. Maddipati, K. Akassoglou, N. Terrando  British Journal of Anaesthesia  Volume 122, Issue 3, Pages 350-360 (March 2019) DOI: 10.1016/j.bja.2018.10.062 Copyright © 2018 The Author(s) Terms and Conditions

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. British Journal of Anaesthesia 2019 122, 350-360DOI: (10.1016/j.bja.2018.10.062) Copyright © 2018 The Author(s) Terms and Conditions

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. British Journal of Anaesthesia 2019 122, 350-360DOI: (10.1016/j.bja.2018.10.062) Copyright © 2018 The Author(s) Terms and Conditions

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. British Journal of Anaesthesia 2019 122, 350-360DOI: (10.1016/j.bja.2018.10.062) Copyright © 2018 The Author(s) Terms and Conditions

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. British Journal of Anaesthesia 2019 122, 350-360DOI: (10.1016/j.bja.2018.10.062) Copyright © 2018 The Author(s) Terms and Conditions

Supplementary Fig S1 Effects of MaR1 on hippocampal gene expression. (A) Filtering data using a statistical significance threshold of P<0.05 and relative expression fold change cutoff of 1.5 vs control mice expression values yielded ∼100 differentially expressed genes, with most of them being down-regulated compared with control values. The heat map shows differentially expressed genes 72 h after surgery (S) and surgery + Maresin 1 (MaR1+S) related to their corresponding control (C) values. Colour code bar indicates control (red) or decreased (shades of green) gene expression fold changes. (B) Analysis of down-regulated genes in the surgery group for the enriched gene ontology terms and biological pathways using David revealed a strong prevalence of genes involved in olfactory sensing, neurological system processes and cognition. n=3 per group for gene array; FC: fold change expression (log2), C: Control, S: Surgery, MaR1: Maresin 1. British Journal of Anaesthesia 2019 122, 350-360DOI: (10.1016/j.bja.2018.10.062) Copyright © 2018 The Author(s) Terms and Conditions

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. British Journal of Anaesthesia 2019 122, 350-360DOI: (10.1016/j.bja.2018.10.062) Copyright © 2018 The Author(s) Terms and Conditions