Role of interleukin-1beta in postoperative cognitive dysfunction

Abstract

Objective: Although postoperative cognitive dysfunction (POCD) often complicates recovery from major surgery, the pathogenic mechanisms remain unknown. We explored whether systemic inflammation, in response to surgical trauma, triggers hippocampal inflammation and subsequent memory impairment, in a mouse model of orthopedic surgery.

Methods: C57BL/6J, knock out (lacking interleukin [IL]-1 receptor, IL-1R(-/-)) and wild type mice underwent surgery of the tibia under general anesthesia. Separate cohorts of animals were tested for memory function with fear conditioning tests, or euthanized at different times to assess levels of systemic and hippocampal cytokines and microglial activation; the effects of interventions, designed to interrupt inflammation (specifically and nonspecifically), were also assessed.

Results: Surgery caused hippocampal-dependent memory impairment that was associated with increased plasma cytokines, as well as reactive microgliosis and IL-1beta transcription and expression in the hippocampus. Nonspecific attenuation of innate immunity with minocycline prevented surgery-induced changes. Functional inhibition of IL-1beta, both in mice pretreated with IL-1 receptor antagonist and in IL-1R(-/-) mice, mitigated the neuroinflammatory effects of surgery and memory dysfunction.

Interpretation: A peripheral surgery-induced innate immune response triggers an IL-1beta-mediated inflammatory process in the hippocampus that underlies memory impairment. This may represent a viable target to interrupt the pathogenesis of postoperative cognitive dysfunction.

FIGURE 1

Surgery-induced systemic inflammation is associated with increased expression of hippocampal interleukin (IL)-1β and is blocked by minocycline. IL-1β and IL-6 levels in plasma were measured by enzyme-linked immunosorbent assay at 2, 6, 24, or 72 hours postintervention. Surgery resulted in increased plasma levels of (A) IL-1β and (B) IL-6 compared to mice receiving the same anesthetics without surgery (Anesthesia) or to naive animals. Administration of minocycline (40mg/kg, intraperitoneally), an antibiotic with anti-inflammatory properties, mitigated surgery-induced elevations in IL-1β and IL-6 in plasma. Enrofloxacin, a antimicrobial comparable to minocycline but devoid of any anti-inflammatory properties, failed to reduce plasma levels of IL-1β, compared to surgical littermates (Surgery) injected with saline (n = 6). (C) Six hours after surgery, IL-1β expression in the hippocampus was increased compared to naive and anesthesia groups. Administration of minocycline but not enrofloxacin mitigated surgery-induced, IL-1β–mediated, hippocampal inflammation (n = 7). Data are expressed as mean ± standard error of the mean. ***p < 0.001; *p < 0.05; for comparison between surgery or enrofloxacin versus naive, anesthetic, and minocycline groups.

FIGURE 2

Surgery induces transcription of interleukin (IL)-1β and IL-6 in the hippocampus. (A) IL-1β and (B) IL-6 mRNA were measured by quantitative real time polymerase chain reaction in hippocampal samples extracted 4, 6, or 24 hours after surgery (S). Naive animals were used as controls. Surgery resulted in increased transcription of both IL-1β and IL-6 in the hippocampus compared to the naive group 6 hours after surgery and had returned to normal by 24 hours after surgery (n = 6). Data are expressed as mean fold change ± standard error of the mean. ***p < 0.001; **p < 0.01; for comparison with naive group.

FIGURE 3

(A–D) Immunohistochemistry of microglia with anti-CD11b. Hippocampi were harvested 1, 3, or 7 days after treatment (pictures shown refer to CA2 region of the hippocampus in tissue harvested after 1 day) and stained with avidin-biotin and 3,3′-diaminobenzidene technique. Representative photomicrographs show (A) naive, (B) anesthetics alone, (C) surgical, and (D) surgical animals treated with minocycline. The amoeboid hypertrophy of cell bodies and clumping of processes seen in the whole hippocampus following surgery are prevented by administration of minocycline. Scale bar = 30μm. (E–G) Densitometry of microglial immunostaining with CD11b. (E) One day after surgery, mice showed significantly higher levels of reactive micro-gliosis compared to naive, anesthetics only, or surgical mice treated with minocycline. (F) Three days after surgery, mice continued to show an increase in reactive microglia compared with naive animals. (G) By 7 days, microglial activation had returned to normal. **p < 0.01 versus naive, anesthesia, and minocycline groups; *p < 0.05 versus naive animals only (n = 7).

FIGURE 4

Hippocampal-dependent recall of fear memories is impaired after surgery. Rodents underwent fear conditioning, and 30 minutes later they were divided to receive anesthetics (Anesthesia), surgery of the tibia under anesthesia (Surgery), or the same surgical procedure with minocycline (Minocycline) or enrofloxacin (Enrofloxacin) administration, respectively. The naive group received no treatment. Contextual and acoustic-cued memories were tested 3 days later. (A) Recall of contextual delay fear conditioning memories was impaired in surgical animals compared to naive and anesthesia groups. Administration of minocycline, but not enrofloxacin, mitigated the surgery-induced decrement in freezing. *p < 0.05 versus naive, anesthesia, and minocycline groups (n = 34). (B) Freezing in the auditory-cued test after delay fear conditioning. There was no difference between the groups in either baseline or auditory cue-related freezing behavior, suggesting that amygdala-dependent memory function is intact after surgery (n = 34). (C) Freezing to context after trace fear conditioning. Mice subjected to surgery exhibited reduced freezing to context when compared to naive animals, confirming that the inflammation induced by surgery disrupts recall of fear contextual memories formed in the hippocampus after trace conditioning (n = 28; *p < 0.05). (D) Hippocampal-dependent, surgery-induced memory impairment is shown in the auditory-cued test, in mice trained with trace fear conditioning. There is a significant difference between the groups in auditory cue-related freezing behavior, suggesting disruption of auditory-cued, hippocampal-dependent retrieval of memories after surgery. No difference was shown in the baseline freezing behavior (n = 28; *p < 0.05). All fear conditioning data are expressed as mean% of time spent freezing ± standard error of the mean.

FIGURE 5

Surgery-induced inflammation is mitigated in mice in which interleukin (IL)-1 signaling is disabled or reduced. (A-B) Immunohistochemistry of hippocampal microglia with anti-CD11b in IL1R^−/− mice. Representative microglia from (A) naive and (B) surgical IL-1R^−/− mice 24 hours after surgery. Scale bar = 30μm. (C) Densitometry of microglial staining in IL1R^−/− mice. Analysis of CD11b immunostaining reveals no difference, confirming that surgery did not activate microglia in IL-1R^−/− mice, compared to untreated littermates, 1 day after the procedure. (D) Circulating IL-1β in IL1R^−/− mice and in wild type pretreated with receptor antagonist (IL-1Ra) prior to surgery. Surgery did not induce a significant increase of IL-1β at 24 hours either in wild type animals treated with IL-1Ra or in IL-1R^−/− mice prior to surgery, but confirmed a significant difference between surgical WT and all other groups. Data are expressed as mean ± standard error of the mean, ***p < 0.001 versus any other group; n = 6. N WT = naive wild type; S WT = wild type undergoing surgery; S WT RA = wild type pretreated with IL-1Ra prior to surgery; S IL1R−/− = mice lacking IL-1R undergoing surgery; N IL1R−/− = naive mice lacking IL-1R.

FIGURE 6

Interleukin-1 receptor antagonist (IL-1Ra) prevents hippocampal neuroinflammation after surgery (S). (A, B) Immunohistochemistry of hippocampal microglia with anti-CD11b in mice pretreated with IL-1Ra. Representative photomicrographs from (A) naive and (B) surgical mice pre-treated with IL-1Ra. Scale bar = 30μm. (C) Densitometry of microglial staining in naive and IL-1Ra–treated surgical mice. (D) Surgery did not activate microglia if IL-1Ra was given preoperatively. Hippocampal expression of IL-1β in IL1-Ra–pretreated surgical mice. Hippocampal IL-1β did not significantly increase in mice treated with IL-1Ra undergoing surgery compared to naive mice. All assessments were conducted 24 hours after surgery. Data are expressed as mean ± standard error of the mean; n = 6.

FIGURE 7

Surgery-induced impairment of contextual fear memories is prevented by preemptive administration of interleukin-1 receptor antagonist (IL-1Ra). (A) IL-1Ra injected before surgery significantly reduced the surgery-induced decrement in freezing behavior. (*p < 0.05 compared to the other groups). (B) Freezing in the auditory-cued test after delay fear conditioning. There was no difference between the groups in either baseline or auditory cue-related freezing behavior, suggesting that neither surgery nor IL-1Ra affected amygdalar-dependent memory function (n = 30). Data are expressed as mean ± standard error of the mean percentage of freezing response.

FIGURE 8

Acquisition of social olfactory memories is not impaired after surgery. The habituation/dishabituation paradigm involved repeated presentations (at 10-minute intervals) of the same stimulus animal, resulting in reduced investigation over successive trials. After 4 successive trials with the same mouse, a novel stimulus animal was presented to rule out fatigue or habituation. In this protocol, the task was combined with a modified discrimination test performed 24 hours after the initial presentation of the first stimulus animal. A previously presented mouse and a new stimulus animal (familiar [Fam]/unfamiliar[Unfam]) were used to test long-term memory. This task returned results showing no difference between naive and surgical mice at any given point of the task, thereby suggesting that, after surgery, specific olfactory memories were normally formed at both 60 minutes and 24 hours. Data are expressed as mean ± standard error of the mean percentage of overall investigation time.