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.

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

In LysM-Cre/Ikkβ^F/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/Ikkβ^F/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/Ikkβ^F/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/Ikk^F/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 Ccr2^RFP/+Cx3cr1^GFP/+ 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<0.01 vs S. On the other hand, administration of PHA 568487 and the other cholinergic agonists were able to rescue the normal phenotype, ** p<0.001 vs S. 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=controls, MLA=methyllycaconitine, PHA=PHA 568487, S=surgery, Nic=nicotine, Chol=choline

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<0.01 vs PHA and control). NF-κB activation was undetectable in unexposed cells (medium) or in cells exposed only to PHA 568487 (PHA). Systemic levels of cytokines were measured by ELISA at different time points; each cohort underwent surgery (tibial fracture under general anesthesia and analgesia) with the exception of naïve controls. Stimulation of the cholinergic signaling pathway with the non-selective BBB permeant (nicotine), impermeant (choline) nAChR agonist, and the selective α7 agonist (PHA) all significantly reduced levels of TNFα (B) and HMGB1 (C) and at all time points (* p<0.01, ** p<0.001 vs S). Delayed production of IL-1β starting from 6 hours following surgery was also reduced by each of the nAChR agonists (D). 24 hours after surgery the nAChR agonists increased levels of anti-inflammatory IL-10, ** p<0.001 vs control (E). Results are expressed as mean ± SEM (n = 5) and compared by one-way analysis of variance followed by Bonferroni's multiple comparison test and Student-Newman-Keuls test. Abbreviations: C=controls, S=surgery, PHA=PHA 568487, Nic=nicotine, Chol=choline, ND=not detected

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, * p<0.05 vs S (A). Conversely, preoperative blockade of cholinergic signaling (MLA+S) exacerbated the deficiency in freezing behavior following surgery. Preoperative administration of nicotine and choline also prevented surgery-induced cognitive decline (* p<0.05, ** p<0.01 vs S, respectively) (B). Mice were treated with MLA or PHA 568487 before undergoing general anesthesia with 2.1% isoflurane for 15 minutes (C). There were no additive effects by cholinergic treatment following exposure to isoflurane anesthesia on memory function 3 days after exposure. Following contextual fear assessment 3 days after surgery, mice were tested on a rotarod to address possible motor impairments and general activity. Fixed speed rotarod: latencies (average of three trials at 15 rpm) were not significantly different between groups, whether exposed to surgical trauma or cholinergic stimulation only (D). Accelerating rotarod: no changes between groups were observed during the accelerating phase (E). Working model (F): damage to the cells following orthopedic surgery promotes rapid release of damage-associated molecular pattern (DAMP) mediators. Within the peripheral macrophages, NF-κB is activated to enhance transcription and subsequent synthesis and release of pro-inflammatory cytokines including TNFα, HMGB1, IL-1 etc, 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