Postoperative analgesia with morphine promoting microglial activation and neuroinflammation induced by surgery aggravates perioperative neurocognitive dysfunction in aged mice

Abstract

Introduction: Perioperative neurocognitive dysfunction (PND) is a significant challenge for patients who need surgery worldwide. Morphine can trigger an intense inflammatory reaction in the central nervous system (CNS) at the same time as analgesia, thus adverse effects aggravating PND. Microglia polarization is closely involved in the regulation of neuroinflammation and the TLR4/MyD88/NF-κB signaling pathway. However, the mechanisms of morphine analgesia aggravating PND impairment remain unclear.

Methods: Tibial fracture surgery was performed in 18 months old male C57BL/6 J mice to mimic human orthopedic surgery and postoperative analgesia with morphine hypodermic or ropivacaine. Levels of inflammatory factors in the hippocampus, activation, and phenotype of microglia, an essential protein of TLR4/MyD88/NF-κB signal pathway, synaptic plasticity, and hippocampal-dependent memory function were evaluated after surgery and postoperative analgesia.

Results: Morphine postoperative analgesia increased the expression of pro-inflammatory cytokines IL-1 β, IL-6, and TNF-α, decreased the level of anti-inflammatory IL-10, aggravated the activation of microglia and the destruction of synaptic plasticity in the hippocampus, resulting in hippocampal neuron loss, a significant decrease in the number of synapses and cognitive impairment in aged mice. In addition, the aggravation of neuroinflammatory response and the activation of microglia may be mediated by TLR4/MyD88/NF- κ B signal pathway.

Conclusion: Our results demonstrate that morphine postoperative analgesia may aggravate microglia activation and neuroinflammation in the hippocampus by regulating the TLR4/MyD88/NF- κ B signal pathway and inhibiting the synaptic plasticity hippocampal neurons. It aggravated the acute cognitive decline and cognitive impairment after tibial fracture in elderly mice.

Keywords: Microglia activation; Morphine; Neuroinflammation; Perioperative neurocognitive dysfunction; Synaptic plasticity.

Fig. 1

Effect of surgery and Morphine postoperative analgesia on aged mice hippocampus inflammation. (A) Timeline for the experimental design. (B) Subjective pain scale. (C) Thermal paw withdrawal latency. (D-G) The level of IL-1β, IL-6, TNF-α, IL-10 in the hippocampus. Data are expressed as means ± SEM (n = 4). *P < 0.05, respectively, compared to the Control group;^#P < 0.05, respectively, compared to the Surgery group;^&P < 0.05, respectively^, compared to the Surgery + Morphine group.

Fig. 2

Effects of surgery and postoperative analgesia on hippocampal neurons from inflammatory cytokines in the aged mice. (A) Representative immunofluorescence images of NeuN-positive cells in the CA1 region. (B) Quantification of NeuN-positive cells in CA1 region. Data are expressed as means ± SEM (n = 4). *P < 0.05, respectively, compared to the Control group;^#P < 0.05, respectively, compared to the Surgery group;^&P < 0.05, respectively^, compared to the Surgery + Morphine group.

Fig. 3

Surgery and postoperative analgesia induced microglial activation in the hippocampus of aged mice. (A) Representative images of Iba1-positive cells in the CA1 region. (B) Quantification of Iba1-positive cells in CA1 region. Data are expressed as means ± SEM (n = 4). *P < 0.05, respectively, compared to the Control group;^#P < 0.05, respectively, compared to the Surgery group;^&P < 0.05, respectively^, compared to the Surgery + Morphine group.

Fig. 4

Role of TLR4/MyD88/NF-κB signaling pathway in microglial activation and neuroinflammation aggravation induced by surgery and postoperative analgesia in aged mice. (A) Representative Western blot bands of Iba1, TLR4, MyD88, and NF-κBp65 in the hippocampus. (B-E) The density analysis results of Iba1, TLR4, MyD88, and NF-κBp65 in the hippocampus. Data are expressed as means ± SEM (n = 4). *P < 0.05, respectively, compared to the Control group;^#P < 0.05, respectively, compared to the Surgery group;^&P < 0.05, respectively^, compared to the Surgery + Morphine group.

Fig. 5

M1 (iNOS, CD86) or M2 (Arg1, CD206) polarization markers are expressed after microglial polarization induced by surgery and postoperative analgesia in aged mice. (A) In the hippocampus, there are representative Western blot brands of iNOS, Arg1, CD86, and CD206. (B-E) The density analysis results of iNOS, Arg1, CD86, and CD206 in the hippocampus. Data are expressed as means ± SEM (n = 4). *P < 0.05, respectively, compared to the Control group;^#P < 0.05, respectively, compared to the Surgery group;^&P < 0.05, respectively^, compared to the Surgery + Morphine group.

Fig. 6

The expression of synaptic plasticity-related proteins (PSD95, SYN) and synaptic number changes after surgery and postoperative analgesia in aged mice. (A) Representative Western blot bands of PSD95 and SYN in the hippocampus. (B-C) The density analysis results of PSD95 and SYN in the hippocampus. (D) The number of synapses in the hippocampus. (E) Electron photomicrographs of synapses in the hippocampus. Data are expressed as means ± SEM (n = 4). *P < 0.05, respectively, compared to the Control group;^#P < 0.05, respectively, compared to the Surgery group;^&P < 0.05, respectively^, compared to the Surgery + Morphine group.

Fig. 7

Surgery and postoperative analgesia induced learning and memory dysfunction in aged mice. (A) Timeline for the experimental design. (B) Freezing time in the fear conditioning test on day 7 after surgery. (C) The representative swimming trace of the aged rats during Morris water maze testing with a hidden platform. (D-E) Escape latency and average swimming speed in the training phase of MWM with a visible platform. (F-G) The number of platform crossings and time spent in the target quadrant in the probe testing of MWM with a hidden platform. Data are expressed as means ± SEM (n = 5). *P < 0.05, respectively, compared to the Control group;^#P < 0.05, respectively, compared to the Surgery group;^&P < 0.05, respectively^, compared to the Surgery + Morphine group.