Stimulation of the α7 nicotinic acetylcholine receptor protects against neuroinflammation after tibia fracture and endotoxemia in mice

Abstract

Surgery and critical illness often associate with cognitive decline. Surgical trauma or infection can lead independently to learning and memory impairments via similar, but not identical, cellular signaling of the innate immune system that promotes neuroinflammation. In this study we explored the putative synergism between aseptic orthopedic surgery and infection, the latter reproduced by postoperative lipopolysaccharide (LPS) administration. We observed that surgery and LPS augmented systemic inflammation up to postoperative d 3 and this was associated with further neuroinflammation (CD11b and CD68 immunoreactivity) in the hippocampus in mice compared with those receiving surgery or LPS alone. Administration of a selective α7 subtype nicotinic acetylcholine receptor (α7 nAChR) agonist 2 h after LPS significantly improved neuroinflammation and hippocampal-dependent memory dysfunction. Modulation of nuclear factor-kappa B (NF-κB) activation in monocytes and regulation of the oxidative stress response through nicotinamide adenine dinucleotide phosphate (NADPH) signaling appear to be key targets in modulating this response. Overall, these results suggest that it may be conceivable to limit and possibly prevent postoperative complications, including cognitive decline and/or infections, through stimulation of the cholinergic antiinflammatory pathway.

Figure 1

Cholinergic stimulation improves cognitive outcome after surgery and LPS administration. (A) Mice were operated on within 30 min after training. At 24 h the S+LPS-group received 1 mg/kg LPS and a bolus injection of PHA 568487 or saline after 2 h. (B) Contextual fear response, as measured by freezing behavior, is impaired in animals receiving surgery followed by LPS exposure as compared with naïve (saline injected) or surgery groups. Administration of a selective α7 nAChR agonist (PHA) significantly improved the cognitive abnormality. (C) Open field was performed after TFC to measure general locomotor activity for a duration of 5 min; no differences in general activity were observed among groups. Data are expressed as mean ± SEM, n = 10. (*P < 0.05 versus C; **P < 0.01 versus C; ^#P < 0.05 versus S; ^&P < 0.05 versus LPS; ^{^^}P < 0.01 versus S+LPS). C, control; S, surgery; PHA, PHA 568487; TFC, trace fear conditioning; OF, open field.

Figure 2

PHA 568487 effects on CD11b activation following surgery and LPS. Hippocampi were harvested at 3 and 7 d after surgery/LPS administration and stained with anti-CD11b. Photomicrographs show CA1 of control animals (C), surgery (S), lipopolysaccharide (LPS), surgery with postoperative LPS (S+LPS) and in combination with a bolus dose of a selective α7 nAChR agonist (S+LPS+PHA) at 3 d. Higher CD11b immunoreactivity was observed after surgery and LPS exposure at both 3 and 7 d (P < 0.01, P < 0.05 respectively). Significant hypertrophy of the cell bodies and loss of pseudopodia occurred in LPS-treated mice after orthopedic surgery; this was attenuated by administration of PHA 568487 2 h after LPS onset (P < 0.01 versus S+LPS). Reactive microglia/monocytes were present up to d 7 in the S+LPS group and returned to baseline in the other treatment groups. Data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls method, n = 4 (**P < 0.01 versus C; ^#P < 0.05 versus S; ^##P < 0.01 versus S; ^&&P < 0.05 versus LPS; ^{^}P < 0.05 versus S+LPS; ^{^^}P > 0.01 versus S+LPS). Scale bar, 50 μm. C, control; S, surgery; PHA, PHA 568487.

Figure 3

Cholinergic agonist reduces reactive macrophages/microglia in the hippocampus after surgery and LPS. Hippocampi were harvested at 3 and 7 d after surgery/LPS administration and stained with anti-CD68. Epifluorescence shows enhanced immunoreactivity 3 d after surgery (S) and lipopolysaccharide (LPS) exposure. Postoperative infection (S+LPS) further increased the number of CD68⁺ cells, persisting up to d 7. Administration of a single bolus dose of a selective α7 nAChR agonist (PHA 568487) significantly reduced levels of CD68⁺ immunoreactivity, returning to baseline at 7 d. Nuclei are counterstained with DAPI. Data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls method, n = 4 (**P < 0.01 versus C; ^#P < 0.05 versus S; ^##P < 0.01 versus S; ^&&P < 0.01 versus LPS; ^{^^}P < 0.01 versus S+LPS). Scale bar, 30 μm. C, control, S, surgery; PHA, PHA 568487.

Figure 4

Infection prolongs systemic and neuroinflammatory response after surgery. Mice were injected with LPS 24 h after orthopedic surgery. Plasma levels of IL-1β and IL-6 were measured by ELISA in plasma and hippocampal tissue. (A) At 3 d following surgery, LPS treated animals had a sustained elevation of IL-1β in plasma. (B) Similarly, systemic levels of IL-6 were also upregulated at 3 d, returning to baseline by 7 d. Data are expressed as mean ± SEM and compared by one-way ANOVA and Student-Newman-Keuls method, n = 4 (**P < 0.01 versus C; ^#P < 0.05 versus S; ^##P < 0.01 versus S; ^&&P < 0.01 versus LPS; ^{^^}P < 0.01 versus S+LPS). C, control; S, surgery.

Figure 5

LPS-induced NF-κB and NADPH activation in BMDMs is blocked by PHA 568487. (A) Cultured BMDMs were stimulated with LPS (100 ng/mL) for 2 h and immunostained for nuclear phosphorylated NF-κB subunit p65. Preincubation with PHA 568487 (10 μg/mL) for 30 min significantly reduced activation and nuclear translocation of p65 (LPS+PHA). NF-κB activation was undetectable in unexposed cells (medium) or in cells exposed to PHA 568487 (PHA). (B) NADPH oxidase mediated superoxide generation from BMDMs was measured by chemiluminescence method. LPS stimulation at 10 ng/mL for 24 h significantly increased superoxide generation from BMDMs (***P < 0.001 versus C), which was abolished by preincubation of PHA (^&&&P < 0.001 versus LPS). PHA per se did not significantly affect superoxide generation in nonstimulated BMDMs. Data are expressed as mean ± SEM and compared by one-way ANOVA and Bonferroni post hoc, n = 6. C, control; PHA, PHA 568487

Figure 6

PHA 568487 impairs cartilage matrix formation. (A) C5.18 cells were differentiated in the presence or absence of 10 μg/mL PHA, and fixed at two time points. (B) Alcian blue staining was performed on whole wells, which were leached and quantified. Data are expressed as mean ± SEM and compared by t test, n = 6. C, control; PHA, PHA 568487.

Figure 7

Working model. Peripheral injury leads to a systemic inflammatory response and release of danger-associated molecular patterns (DAMPs) in the circulation causing neuroinflammation and subsequent cognitive decline. Postoperative infection elicits a secondary hit that exacerbates the proinflammatory milieu and CNS dysfunction. Stimulation of the cholinergic antiinflammatory signaling pathway via selective α7 nAChR improves cognitive decline though modulation of NF-κB and oxidative stress activity on monocytes, thus attenuating neuroinflammation and dampening the systemic proinflammatory milieu.