Critical role of P2X7 receptors in the neuroinflammation and cognitive dysfunction after surgery

Abstract

Postoperative cognitive dysfunction worsens patient outcome after surgery. Neuroinflammation is a critical neuropathological process for it. We determined the role of P2X7 receptors, proteins that participate in inflammatory response, in the neuroinflammation induction after surgery, and whether the choice of volatile anesthetics affects its occurrence. Eight-week old C57BL/6J or P2X7 receptor knockout male mice were subjected to right carotid arterial exposure under anesthesia with 1.8% isoflurane, 2.5% sevoflurane or 10% desflurane. They were tested by Barnes maze and fear conditioning from 2weeks after the surgery. Hippocampus was harvested 6h, 24h and 7days after the surgery for immunohistochemical staining and Western blotting. Mice with surgery under anesthesia with isoflurane, sevoflurane or desflurane took longer than control mice to identify the target box 1 or 8days after the training sessions in Barnes maze. Mice anesthetized by isoflurane or sevoflurane, but not by desflurane, had less freezing behavior than control mice in fear conditioning test. Mice with surgery and anesthesia had increased ionized calcium binding adapter molecule 1 and interleukin 1β in the hippocampus but this increase was smaller in mice anesthetized with desflurane than mice anesthetized with isoflurane. Mice with surgery had increased P2X7 receptors and its downstream molecule caspase 1. Inhibition or knockout of P2X7 receptors attenuated surgery and anesthesia-induced neuroinflammation and cognitive impairment. We conclude that surgery under desflurane anesthesia may have reduced neuroinflammation and cognitive impairment compared with surgery under isoflurane anesthesia. P2X7 receptors may mediate the neuroinflammation and cognitive impairment after surgery.

Keywords: Learning and memory; Neuroinflammation; P2X7 receptors; Volatile anesthetics.

Fig. 1

Surgery and anesthesia impaired the learning and memory. Mice were started to be tested by Barnes maze and fear conditioning 2 weeks after the surgery. A: training sessions of Barnes maze. Results are mean ± S.E.M. (n = 12 – 13). * P < 0.05 compared with the corresponding data on day 1. B: memory phase of Barnes maze. Results are in box plot format (n = 12 – 13). •: lowest or highest score (the score will not show up if it falls in the 95th percentile); between lines: 95th percentile of the data; inside boxes: 25th to 75th percentile including the median of the data. ^ P < 0.05 compared with control group. C: Fear conditioning. Results are mean ± S.E.M. (n = 12 – 13). ^ P < 0.05 compared with control group; # P < 0.05 compared with surgery plus isoflurane anesthesia.

Fig. 2

Surgery and anesthesia induced neuroinflammation. Hippocampus was harvested 6 h after the surgery. A: representative images of Iba-1 staining. Scale bar = 100 µm. B: quantitative data of Iba-1. C: IL-1β concentrations in the hippocampus. Results are mean ± S.E.M. (n = 6). * P < 0.05 compared with control group; ^ P < 0.05 compared with surgery plus isoflurane anesthesia.

Fig. 3

Surgery increased P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h after the surgery for immunohistochemical staining or 24 h after the surgery for Western blotting. A: representative images of immunostaining. Scale bar = 100 µm. B: representative images of Western blotting. C: quantitative data of Western blotting. Results are mean ± S.E.M. (n = 5 – 6). * P < 0.05 compared with control group.

Fig. 4

Inhibition of P2X7 receptors attenuated surgery-induced learning and memory impairment. Mice were started to be tested by Barnes maze and fear conditioning 2 weeks after the surgery under isoflurane anesthesia. A: training sessions of Barnes maze. Results are mean ± S.E.M. (n = 12). * P < 0.05 compared with the corresponding data on day 1. B: memory phase of Barnes maze. Results are in box plot format (n = 12). •: lowest or highest score (the score will not show up if it falls in the 95th percentile); between lines: 95th percentile of the data; inside boxes: 25th to 75th percentile including the median of the data. ^ P < 0.05 compared with control group. # P < 0.05 compared with surgery plus isoflurane anesthesia group. C: Fear conditioning. Results are mean ± S.E.M. (n = 11 – 12). ^ P < 0.05 compared with control group; # P < 0.05 compared with surgery plus isoflurane anesthesia.

Fig. 5

Inhibition of P2X7 receptors attenuated surgery-induced neuroinflammation. Hippocampus was harvested 6 h or 7 days after the surgery. A: representative images of Iba-1 staining of hippocampus harvested 6 h after the surgery. Scale bar = 100 µm. B: quantitative data of Iba-1 of hippocampus harvested 6 h after the surgery. C: representative images of Iba-1 staining of hippocampus harvested 7 days after the surgery. D: quantitative data of Iba-1 of hippocampus harvested 7 days after the surgery. Results are mean ± S.E.M. (n = 5 for panels B and D). * P < 0.05 compared with control group; ^ P < 0.05 compared with surgery plus isoflurane anesthesia group.

Fig. 6

Inhibition of P2X7 receptors attenuated surgery-induced increase of P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h or 7 days after the surgery under isoflurane anesthesia. A and B: representative images of Western blotting. C: quantitative data of P2X7 receptors. D: quantitative data of P20. E: quantitative data of precursor caspase 1. Results are mean ± S.E.M. (n = 6). * P < 0.05 compared with control group; ^ P < 0.05 compared with the corresponding surgery plus isoflurane anesthesia group; # P < 0.05 compared with the level of P20 at 6 h after surgery under isoflurane anesthesia.

Fig. 7

P2X7 receptor knockout blocked surgery-induced neuroinflammation and cognitive impairment. Cerebral cortex and hippocampus of C57BL/6J mice or P2X7 receptor knockout mice were harvested for Western blotting of P2X7 receptors. Also, P2X7 receptor knockout mice were started to be tested by Barnes maze and fear conditioning 2 weeks after the surgery under isoflurane anesthesia. Mouse hippocampus was harvested 6 h after the surgery for measuring IL-1β. A: Western blot images of P2X7 receptor. B: training sessions of Barnes maze. Results are mean ± S.E.M. (n = 8). * P < 0.05 compared with the corresponding data on day 1. C: memory phase of Barnes maze. Results are in box plot format (n = 8). •: lowest or highest score (the score will not show up if it falls in the 95th percentile); between lines: 95th percentile of the data; inside boxes: 25th to 75th percentile including the median of the data. D: Fear conditioning. Results are mean ± S.E.M. (n = 8). E: IL-1β concentrations in the hippocampus. Results are mean ± S.E.M. (n = 5).