Dysfunction of inflammation-resolving pathways is associated with exaggerated postoperative cognitive decline in a rat model of the metabolic syndrome

Abstract

The cholinergic antiinflammatory pathway (CAP), which terminates in the spleen, attenuates postoperative cognitive decline (PCD) in rodents. Surgical patients with metabolic syndrome exhibit exaggerated and persistent PCD that is reproduced in postoperative rats selectively bred for easy fatigability and that contain all features of metabolic syndrome (low-capacity runners [LCRs]). We compared the CAP and lipoxin A(4) (LXA(4)), another inflammation-resolving pathway in LCR, with its counterpart high-capacity runner (HCR) rats. Isoflurane-anesthetized LCR and HCR rats either underwent aseptic trauma involving tibial fracture (surgery) or not (sham). At postoperative d 3 (POD3), compared with HCR, LCR rats exhibited significantly exaggerated PCD (trace fear conditioning freezing time 43% versus 57%). Separate cohorts were killed at POD3 to collect plasma for LXA4 and to isolate splenic mononuclear cells (MNCs) to analyze CAP signaling, regulatory T cells (Tregs) and M2 macrophages (M2 Mφ). Under lipopolysaccharide (LPS) stimulation, tumor necrosis factor (TNF)-α produced by splenic MNCs was 117% higher in LCR sham and 52% higher in LCR surgery compared with HCR sham and surgery rats; LPS-stimulated TNF-α production could not be inhibited by an α7 nicotinic acetylcholine receptor agonist, whereas inhibition by the β(2) adrenergic agonist, salmeterol, was significantly less (-35%) than that obtained in HCR rats. Compared to HCR, sham and surgery LCR rats had reduced β(2) adrenergic receptor-expressing T lymphocytes (59%, 44%), Tregs (47%, 54%) and M2 Mφ (45%, 39%); surgical LCR rats' hippocampal M2 Mφ was 66% reduced, and plasma LXA4 was decreased by 120%. Rats with the metabolic syndrome have ineffective inflammation-resolving mechanisms that represent plausible reasons for the exaggerated and persistent PCD.

Figures

Figure 1

Postoperative neuroinflammatory response to peripheral aseptic trauma. Aseptic surgical trauma induces binding of damage-associated molecular patterns (DAMPs) to pattern recognition receptors (PRR); this engages the innate immune system via NF-κB–dependent signaling in monocytes, to synthesize and release proinflammatory cytokines, including TNF-α, which disrupts the blood brain barrier (BBB) (–8). Through a permeable BBB, CCR2-expressing bone marrow–derived macrophages (BM-DM) are attracted by the newly expressed chemokine MCP-1 into the brain parenchyma (5). Within the hippocampus, the activated macrophages release proinflammatory cytokines that are capable of disrupting long-term potentiation, the neurobiologic correlate of learning and memory (,–11).

Figure 2

Cholinergic inflammation-resolving pathway (–15). At its splenic nerve terminus, vagal outflow releases adrenergic agonists (rather than the usual cholinergic neuro-transmitter); these catecholamines (CA) activate β2 ARs on CD3 T lymphocytes that contain choline acetyltransferase (CHAT), which is capable of synthesizing the acetylcholine needed to mediate inhibition of macrophage NF-κB activity by signaling through α7 nAChR.

Figure 3

Postoperative cognitive decline is exaggerated in LCR rats coincident with time when splenic and peripheral MNCs in the LCR rats are more proinflammatory. (A) Freezing percentage in trace fear conditioning in LCR and HCR rats under sham and surgical conditions at d 3; #p < 0.05 HCR surgery versus LCR surgery. (B) TNF-α levels were higher in the media of cultured LCR splenic mononuclear cells without LPS challenge; **p < 0.01 HCR versus LCR. (C) TNF-α levels were reduced in the media of cultured HCR blood mononuclear cells stimulated with LPS (1 μmol/L); *p < 0.05 HCR sham versus LCR sham; #p < 0.05 HCR surgery versus LCR surgery at d 3. (D) TNF-α levels were decreased in the media of cultured HCR splenic mononuclear cells stimulated with LPS; **p < 0.01 HCR sham versus LCR sham; #p < 0.05 HCR surgery versus LCR surgery at d 3. Each data point represents one independent experiment. Values are means ± SD. TFC, trace fear conditioning.

Figure 4

In response to LPS and surgery challenges, impairment of the cholinergic α7 nAChR pathway in LCR rats enhanced proinflammatory responses in splenic MNCs compared with HCR rats. (A) Decreased cAMP levels in LCR splenic MNCs stimulated with LPS (1 μmol/L); **p < 0.01 HCR compared with LCR. (B) Less inhibitory effects of nicotine on TNF-α levels in LCR splenic MNCs stimulated with LPS; **p < 0.01 HCR compared with LCR. (C) Less inhibitory effects of PHA 568487 (PHA, 1 μmol/L) on p65 NF-κB levels in LCR splenic MNCs stimulated with LPS; **p < 0.01 HCR compared with LCR in PHA 568487 pretreated group; n = 3 in each group; MLA (1 μmol/L) counteracted the inhibitory effects of PHA 568487. (D) Less inhibitory effects of salmeterol on TNF-α levels in LCR splenic MNCs stimulated with LPS; **p < 0.01 HCR compared with LCR in the salmeterol pretreated group. (E) Less effect of salmeterol (1 μmol/L) on increasing IL-10 levels in LCR MNCs stimulated with LPS; **p < 0.01 HCR compared with LCR in the salmeterol pretreated group. Each data point represents one independent experiment. Values are means ± SD.

Figure 5

M1 macrophages were elevated and proresolving α7 nAChR+CD11b/c+ cells were decreased in the spleen of LCR rats. (A) CD11b/c was used as a marker to analyze M1 macrophages splenocytes by flow cytometry in LCR and HCR rats under sham and surgical conditions at d 3. (B) Absolute number of M1 macrophages in each spleen was calculated by multiplying the percentage of CD11b/c+ cells by total number of splenocytes at d 3; n = 3 in each group. **p = 0.01 versus LCR sham. (C) Flow cytometry was used to analyze α7 nAChR+CD11b/c+ cells in the splenocytes. Values are means ± SD.

Figure 6

Reduction of β2 AR–expressing T lymphocytes, regulatory T cells and impairment of M2 macrophage polarization in LCR rats. (A) Analysis of β2 AR expression in splenic lymphocytes by flow cytometry in LCR and HCR rats under sham and surgical conditions; n = 3–6 in each group; **p < 0.01; #p < 0.05. (B) Changes of regulatory T cells in splenic lymphocytes in LCR and HCR rats under sham and surgical conditions; n = 3–6 in each group; *p < 0.01; #p < 0.05. (C) Arginase 1 was used as marker to analyze M2 macrophages in splenocytes by flow cytometry in LCR and HCR rats under sham and surgical conditions; n = 3–6 in each group; *p < 0.01; #p < 0.05. Each data point represents one independent experiment. p values are shown. Values are means ± SD.

Figure 7

Changes of proinflammatory- and inflammation-resolving mechanisms in the hippocampus of LCR rats after surgery. (A) IL-6 mRNA levels in hippocampus in LCR and HCR rats under sham and surgical conditions at postoperative d 3; *p < 0.05 HCR surgery versus LCR surgery. (B) The percentage of M2 macrophages in the hippocampus in LCR and HCR rats under sham and surgical conditions at d 3; ##p < 0.01 HCR surgery versus LCR surgery; each data point represents one independent experiment. Values are means ± SD.

Figure 8

Defects of metabolism of eicosanoids in the LCR rats after surgery caused imbalance of plasma LXA4 and LTB4. (A) Changes of plasma LXA4 among four groups of rats. **p < 0.01 for LCR sham versus LCR surgery; ##p < 0.01 for HCR sham versus HCR surgery; ¶p < 0.05 for LCR surgery versus HCR surgery; n = 8 in each group. (B) Changes of plasma LTB4 among four groups of rats. **p < 0.01 for LCR sham versus LCR surgery; #p < 0.05 for HCR sham versus HCR surgery; ¶¶p < 0.01 for LCR surgery versus HCR surgery; n = 4 in each group. Each data point represents one independent experiment. Values are means ± SD.