Resolving postoperative neuroinflammation and cognitive decline

Abstract

Objective: Cognitive decline accompanies acute illness and surgery, especially in the elderly. Surgery engages the innate immune system that launches a systemic inflammatory response that, if unchecked, can cause multiple organ dysfunction. We sought to understand the mechanisms whereby the brain is targeted by the inflammatory response and how this can be resolved.

Methods: C57BL/6J, Ccr2(RFP/+)Cx3cr1(GFP/+), Ikk(F/F) mice and LysM-Cre/Ikk(F/F) mice underwent stabilized tibial fracture operation under analgesia and general anesthesia. Separate cohorts of mice were tested for systemic and hippocampal inflammation, integrity of the blood-brain barrier (BBB), and cognition. The putative resolving effects of the cholinergic pathway on these postoperative responses were also studied.

Results: Peripheral surgery disrupts the BBB via release of tumor necrosis factor-alpha (TNFα), which facilitates the migration of macrophages into the hippocampus. Macrophage-specific deletion of Ikappa B kinase (IKK)β, a central coordinator of TNFα signaling through activation of nuclear factor (NF) κB, prevents BBB disruption and macrophage infiltration in the hippocampus following surgery. Activation of the α7 subtype of nicotinic acetylcholine receptors, an endogenous inflammation-resolving pathway, prevents TNFα-induced NF-κB activation, macrophage migration into the hippocampus, and cognitive decline following surgery.

Interpretation: These data reveal the mechanisms for bidirectional communication between the brain and immune system following aseptic trauma. Pivotal molecular mechanisms can be targeted to prevent and/or resolve postoperative neuroinflammation and cognitive decline.

Copyright © 2011 American Neurological Association.

Figures

Figure 1. NF-κB activation in myeloid cells mediates BBB disruption and macrophage migration into the hippocampus following surgery

In LysM-Cre/IkkβF/F mice surgery produced neither CD11b immunoreactivity (A) as measured with the avidin-biotin and 3,3’-diaminobenzidene technique, nor signs of peripheral inflammation (B) as reflected by normal levels of TNFα and IL-1β (* p<0.05, ** p<0.01 vs LysM-Cre/IkkβF/F+S). Compared to naïve animals (c) surgery increased expression of immunolabeled fibrinogen in the hippocampus at 24 h postoperatively, *** p<0.001 (C). LysM-Cre/IkkβF/F had no fibrinogen deposition in the brain compared to operated wild-type mice. Immunolabeling for fibrinogen shows significant deposition in the hippocampus 1 day following surgery. Perivascular fibrinogen was also significantly elevated following MLA administration and surgery (MLA+S). Notably, quantification revealed that control mice, surgical mice treated with PHA 568487 and LysM-Cre/IkkF/F had no fibrinogen deposition in the brain (C). Data are expressed as mean ± SEM (n = 4) and compared by one-way analysis of variance and Student-Newman-Keuls method. Scale bars: 30μm. Abbreviations: C=control, MLA=methyllycaconitine, PHA=PHA 568487, S=surgery.

Figure 2. Surgery-induced neuroinflammation associates with migration of peripheral macrophages

Epifluorescence of Ccr2RFP/+Cx3cr1GFP/+ shows no RFP+ cells in controls (c) or surgical animals treated with the selective α7 agonist (PHA+S) or anti-TNF antibody (antiTNF+S). One day after surgery a significant increase in RFP+ cells was observed around the periventricular lining into and into the parenchyma of the hippocampus (S), but not in other brain regions. Blockade of the cholinergic pathway exacerbated neuroinflammation and infiltration of macrophages in the hippocampus, * p<0.05 (MLA+S). Resident microglia (CX3CR1) maintained long and ramified pseudopodia in all groups, suggesting that this subset of resident cells was not involved in surgery induced neuroinflammation or its modulation by nAChRs. Data are expressed as mean ± SEM (n = 4) and compared by one-way analysis of variance and Student-Newman-Keuls method. Abbreviations: C=controls, S=surgery, MLA=methyllycaconitine, PHA=PHA 568487

Figure 3. Effects of cholinergic modulation on neuroinflammation

Hippocampi were extracted 1 day after surgery and CD11b immunoreactivity was measured with the avidin-biotin and 3,3’-diaminobenzidene technique. Representative photomicrographs show control animals, tibial surgery under general anesthesia and analgesia (S), surgery with preoperative administration of either a bolus dose of a nAChR antagonist (MLA+S), a selective α7 agonist (PHA+S), nicotine (Nic+S) or choline (Chol+S). Densitometry of CD11b immunostaining revealed significant higher immunoreactivity as characterized by hypertrophy of cell bodies and clumping of the ramifications following surgery and surgery in the presence of the cholinergic antagonist, * p

Figure 4. Stimulation of α7 nAChR blocks TNF-induced NF-κB activation in BMDMs in vitro and postoperative systemic inflammation

Bone marrow was extracted from long bones of the hind limbs of C57BL6/J mice and BMDMs isolated and differentiated for 7 days (A). Cells were stimulated with TNFα (40 ng/ml) for 2 h and immunostained for nuclear phosphorylated NF-κB subunit p65, this was significantly attenuated when pre-incubated with PHA 568487 (10 μg/ml) for 30 min (TNF+PHA) (* p

Figure 5. Effects of cholinergic modulation on memory function following surgery

Contextual fear response reveals hippocampal-dependent memory impairment at postoperative day 3. Preoperative treatment with a selective α7nAChR agonist (PHA+S) prevented postoperative memory impairment, * petc, further augmenting NF-κB transcription via activation of different receptors (PRRs). Systemic cytokines, in particular TNFα, augment BBB permeability, ultimately allowing peripheral immunocompetent cells to invade the CNS. Macrophages once in the brain parenchyma account for the neuroinflammatory changes, affecting hippocampal networks, synapses and neurons and ultimately affecting memory function. Cholinergic agonists activate the nAChR on peripheral macrophages to inhibit NF-κB-induced inflammation, BBB disruption and postoperative cognitive decline. Data are expressed as mean ± SEM (n = 10) and compared by one-way or two-way (rotarod) analysis of variance and Student-Newman-Keuls method. Abbreviations: N=naïve, MLA=methyllycaconitine, PHA=PHA 568487, S=surgery, Nic=nicotine, Chol=choline