Orthopedic surgery-induced cognitive dysfunction is mediated by CX3CL1/R1 signaling

Abstract

Background: Postoperative pain is a common phenomenon after surgery and is closely associated with the development of postoperative cognitive dysfunction (POCD). Persistent pain and systemic inflammation caused by surgery have been suggested as key factors for the development of POCD. Fractalkine (CX3CL1) and its receptor, the CX3C chemokine receptor 1 (CX3CR1), are known to play a key role in pain and inflammation signaling pathways. Recent studies have shown that the regulation of CX3CR1/L1 signaling influences the development of various diseases including neuronal diseases. We determined whether CX3CR1/L1 signaling is a putative therapeutic target for POCD in a mouse model.

Methods: Adult (9-11 weeks) male mice were treated with neutralizing antibody to block CX3CR1/L1 signaling both before and after surgery. Inflammatory and behavioral responses including pain were assessed postoperatively. Also, CX3CR1 mRNA level was assessed. Hippocampal astrocyte activation, Mao B expression, and GABA expression were assessed at 2 days after surgery following neutralizing antibody administration.

Results: The behavioral response indicated cognitive dysfunction and development of pain in the surgery group compared with the control group. Also, increased levels of pro-inflammatory cytokines and CX3CR1 mRNA were observed in the surgery group. In addition, increased levels of GABA and increased Mao B expression were observed in reactive astrocytes in the surgery group; these responses were attenuated by neutralizing antibody administration.

Conclusions: Increased CX3CR1 after surgery is both necessary and sufficient to induce cognitive dysfunction. CX3CR1 could be an important target for therapeutic strategies to prevent the development of POCD.

Keywords: CX3C chemokine receptor 1; GABA; Hippocampus; Inflammation; Postoperative cognitive dysfunction; Postoperative pain.

Fig. 1

Schematic diagram of the experimental procedure. In experiment 1, the mice underwent behavioral tests for the training phase 3 h before surgery and post behavioral tests 1, 2, and 5 days after surgery. Brain tissues were immediately harvested at the end of the behavioral testing, and ELISA analysis for TNF-α, IL-6, and IL-1β levels was performed. The level of CX3CR1 mRNA was measured by RT-PCR. In experiment 2, neutralizing Ab was injected 30 min before TF surgery after behavioral training tests, and at the same time point 24 and 48 h after later. The mice performed behavioral tests for the training phase 3 h before surgery and post behavioral tests 2 days after surgery. The brain tissues were harvested and ELISA and immunohistochemistry was performed after the behavioral testing period. For OIS imaging, the mice received the cranial window implantation 3 weeks before surgery, and OIS imaging was performed 2 days after surgery

Fig. 2

Assessment of neurobehavioral abilities of POCD mice. a Total distance traveled and b percentage of time in center zone in the open-field test. c Latency to enter the dark compartment in the passive avoidance test. d Learning index (entering time in which a mouse first moves from the open arms to closed arm during the training period − entering time in which a mouse first moves from the open arms to closed arm during the test period) in the elevated plus maze test. e Discrimination time rate for novel objects in the test phase of the novel objective recognition test (unpaired t-test, n = 8~11 per group). f Mechanical allodynia was performed using the von Frey test (two-way ANOVA with Tukey’s post hoc test, n = 15 per group). Con, normal control group; TF, tibial fracture surgery group; 1D, 1 day after surgery; 2D, 2 days after surgery; 5D, 5 days after surgery; AP, acquisition phase; TP, test phase; *p < 0.05 compared to the control; **p < 0.01 compared to the control

Fig. 3

The level of pro-inflammatory cytokines and mRNA expression in POCD mice. a IL-1β, IL-6, and TNF-α in the prefrontal cortex, the hippocampus, and the amygdala at different time points after tibial fracture surgery (one-way ANOVA with Tukey’s post hoc, n = 8~11 per group). b The mRNA level of CX3CR1 in the prefrontal cortex, the hippocampus, and the amygdala at different time points after tibial fracture surgery (one-way ANOVA with Tukey’s post hoc, n = 4~5 per group). Control, normal control group; TF, tibial fracture surgery group; 2hr, 2 h after surgery; 6hr, 6 h after surgery; 24hr, 1 day after surgery; 2D, 2 days after surgery; 5D, 5 days after surgery; *p < 0.05 compared to the control

Fig. 4

Evaluation of neurobehavioral abilities of POCD mice after inhibition of CX3CR1 signaling (one-way ANOVA with Tukey’s post hoc, n = 5~13 per group). a Latency to enter the dark compartment in the passive avoidance test 2 days after tibial fracture surgery. b Leaning index (transfer latency time to enter the closed arm of the training period − transfer latency time to enter the closed arm during the test period) in the elevated plus maze test. c Discrimination time rate for novel objects in the test phase of the novel objective recognition test. d Time rate in the center zone in the open-field test. e Mechanical allodynia was evaluated with the von Frey test. Control, normal control group; TF2D, 2 days after tibial fracture surgery group; TF+Ab, 2 days after tibial fracture surgery group injected with neutralizing Ab; Control+Ab, normal control group injected with neutralizing Ab; *p < 0.05 compared to the control; ^#p < 0.05 compared to the TF2D; ^###p < 0.0001 compared to TF2D

Fig. 5

Anti-inflammatory effect of inhibition of CX3CR1 signaling in tibial fracture-induced POCD mice. The levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the prefrontal cortex, the hippocampus, and the amygdala (one-way ANOVA with Tukey’s post hoc, n = 3~10 per group). Control, normal control group; TF2D, 2 days after tibial fracture surgery group; TF+Ab, 2 days after tibial fracture surgery group injected with neutralizing Ab; Control+Ab, normal control group injected with neutralizing Ab. *p < 0.05; **p < 0.01; ***p < 0.0001 compared to the control; ^#p < 0.05; ^##p < 0.01; ^###p < 0.0001 compared to TF2D

Fig. 6

Impaired neurovascular coupling response to sensory stimulation in POCD. a Representative image of the intensity changes of the optical imaging signal during whisker stimulation. b Time course traces of relative changes in cerebral blood volume after stimulation. c Average of the maximum cerebral blood volume response (one-way ANOVA with Tukey’s post hoc test, n = 4 or 6 per group). Control, normal control group; TF2D, 2 days after tibial fracture surgery group; TF+Ab, 2 days after tibial fracture surgery group injected with neutralizing Ab. **p < 0.01 compared to the control; ^##p < 0.01 compared to TF2D

Fig. 7

Effect of inhibition of CX3CR1 signaling on GFAP, Mao B expression, and GABA levels. a Representative immunofluorescence images for DAPI, GFAP, and Mao B. b Sholl analysis for a traced individual GFAP-positive astrocyte, which is superimposed over concentric circles (one-way ANOVA with Tukey’s post hoc test, n = 8 per group). c Intensity of Mao B images. d Quantification of the total number of intercepts with ramifications from Sholl analysis (one-way ANOVA with Tukey’s post hoc test, n = 10 per group). a Representative immunofluorescence images for DAPI and GABA. b Quantification of GABA-positive cells (one-way ANOVA with Tukey’s post hoc test, n = 8 per group). Control, normal control group; TF_Veh, 2 days after tibial fracture surgery group injected with saline; TF_Ab, 2 days after tibial fracture surgery group injected with neutralizing Ab; Control+Ab, normal control group injected with neutralizing Ab; scale bar, 20 μm, 100 μm. *p < 0.05; **p < 0.01; ***p < 0.0001 compared to the control; ^#p < 0.05; ^##p < 0.01; ^###p < 0.0001 compared to TF2D