CCL2 Inhibitor Bindarit Improve Postoperative Cognitive Function by Attenuating Pericyte Loss-Related Blood-Brain Barrier Disruption and Neuroinflammation

Abstract

Perioperative neurocognitive disorder (PND) is a common complication in elderly patients undergoing surgery and anesthesia and associated with the impaired recovery. Previous studies have demonstrated that PND was correlated with the pericyte (PC) loss in brain, but the underlying mechanisms remain unclear. This study investigates whether C-C motif chemokine ligand 2 (CCL2) in hippocampal tissues contributes to postoperative PC injury, blood-brain barrier (BBB) disruption, neuroinflammation, and cognitive dysfunction. Sixteen-month-old C57BL/6 mice underwent tibial fracture surgery to induce PND. CCL2 expression in hippocampal tissues was downregulated using intraperitoneal injections of 200 mg/kg daily Bindarit for 4 days prior to surgery. Behavioral tests were conducted on the third day postsurgery and brain tissues were collected. Western blotting assessed CCL2 expression in the hippocampus, immunofluorescence evaluated PC coverage, BBB integrity, and neuroinflammation, and transmission electron microscopy (TEM) examined BBB microstructure. Bindarit effectively inhibited the surgery-induced increase in hippocampal CCL2 expression and improved postoperative cognitive function. Behavioral tests, including the open field (OF) test and novel object recognition (NOR) test, indicated enhanced locomotor activity and short-term memory in Bindarit-treated mice compared to controls. Immunofluorescence analysis revealed that Bindarit treatment mitigated the reduction in capillary length and tight junction (TJ) protein expression, specifically claudin-5 and occludin, which was seen with decreased PC coverage. Additionally, Bindarit suppressed the activation of hippocampal microglia and astrocytes, as evidenced by reduced Iba-1 and GFAP staining. TEM analysis confirmed that Bindarit preserved BBB microstructure integrity postsurgery. This study demonstrates that the CCL2 inhibitor Bindarit significantly reduces the incidence of PND by preventing PC loss, thereby protecting the BBB and alleviating neuroinflammation. These findings suggest that targeting CCL2 could be a potential therapeutic strategy for preventing and treating PND.

Keywords: CCL2; blood–brain barrier; neuroinflammation; pericyte; perioperative neurocognitive disorder.

Figure 1

Schematic diagram of the experimental procedures. The Bindarit group received intraperitoneal injections of Bindarit (20 mg/kg) daily for 4 days prior to surgery. Postsurgery, one set of mice was sacrificed on Day 3 for biochemical analysis. A separate set of mice underwent behavioral tests: the open field (OF) test on Day 3 to assess locomotor activity and anxiety, the novel object recognition (NOR) test on Days 4 and 5 to evaluate short-term memory, and the fear conditioning (FC) test on Days 6 and 7 to assess contextual and cued fear memory.

Figure 2

Bindarit reduced CCL2 expression in the hippocampus after anesthesia/surgery. (A) Immunoblotting of CCL2 expression in hippocampus. (B) Quantitative analysis of gray value of CCL2 immunoblotting. Data are presented as mean ± SEM (n = 3), ⁣^∗∗∗p  < 0.001.

Figure 3

Bindarit alleviated anesthesia/surgery induced cognitive impairment. (A) Representative images of motion trajectories in the OF test. (B) Average speed of mice in the OF test. (C) Activity time of mice in the central area during the OF test. (D) Distance moved by mice in the central area during the OF test. (E) Representative images of motion trajectories in the NOR test. (F) RI of mice in the NOR test. (G) Freezing time during contextual fear memory test. (H) Freezing time during cued fear memory test. Data are presented as mean ± SEM (n = 7), ⁣^∗p  < 0.05, ⁣^∗∗p  < 0.01, ⁣^∗∗∗p  < 0.001.

Figure 4

Bindarit inhibited the loss of PC and reduction of capillary length induced by anesthesia/surgery. (A) Representative confocal images of CD13-positive PC (red) and lectin-positive capillary (green) in the mice hippocampus. (B) Quantitative analysis of CD13-positive PC coverage. (C) Quantitative analysis of lectin-positive capillary length. (D) Correlation analysis between PC coverage and capillary length. Data are presented as mean ± SEM (n = 6), ⁣^∗p  < 0.05, ⁣^∗∗p  < 0.01, ⁣^∗∗∗p  < 0.001.

Figure 5

Bindarit reduced the decrease of claudin-5 induced by anesthesia/surgery. (A) Representative confocal images of claudin-5 (red) and lectin-positive capillary (green) in the mice hippocampus. (B) Quantitative analysis of claudin-5 length. (C) Correlation analysis between PC coverage and claudin-5 length. Data are presented as mean ± SEM (n = 6), ⁣^∗p  < 0.05, ⁣^∗∗∗p  < 0.001.

Figure 6

Bindarit reduced the decrease of occludin induced by anesthesia/surgery. (A) Representative confocal images of occludin (red) and lectin-positive capillary (green) in the mice hippocampus. (B) Quantitative analysis of occludin length. (C) Correlation analysis between PC coverage and occludin length. Data are presented as mean ± SEM (n = 6), ⁣^∗p  < 0.05, ⁣^∗∗∗p  < 0.001.

Figure 7

Bindarit alleviated BBB ultrastructure damage induced by anesthesia/surgery. Representative images of hippocampal ultrastructure under TEM in each group of mice, the arrows indicate the TJ.

Figure 8

Bindarit reduced the enhanced activation of microglia and astrocytes induced by anesthesia/surgery. (A) Representative confocal images of Iba-1-positive microglia and GFAP-positive astrocytes in the hippocampal CA1 region of mice. (B) Quantitative percentage of the area occupied by Iba-1-positive microglia. (C) Correlation analysis between PC coverage and the activated area of Iba-1-positive microglia. (D) Quantitative percentage of the area occupied by GFAP-positive astrocytes. (E) Correlation analysis between PC coverage and the activated area of GFAP-positive astrocytes. Data are presented as mean ± SEM (n = 6), ⁣^∗p  < 0.05, ⁣^∗∗p  < 0.01, ⁣^∗∗∗p  < 0.001.