NLRP3-GABA signaling pathway contributes to the pathogenesis of impulsive-like behaviors and cognitive deficits in aged mice

Abstract

Background: Perioperative neurocognitive disorders (PND), such as delirium and cognitive impairment, are commonly encountered complications in aged patients. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is aberrantly synthesized from reactive astrocytes following inflammatory stimulation and is implicated in the pathophysiology of neurodegenerative diseases. Additionally, the activation of NOD-like receptor protein 3 (NLRP3) inflammasome is involved in PND. Herein, we aimed to investigate whether the NLRP3-GABA signaling pathway contributes to the pathogenesis of aging mice's PND.

Methods: 24-month-old C57BL/6 and astrocyte-specific NLRP3 knockout male mice were used to establish a PND model via tibial fracture surgery. The monoamine oxidase-B (MAOB) inhibitor selegiline (1 mg/kg) was intraperitoneally administered once a day for 7 days after the surgery. PND, including impulsive-like behaviors and cognitive impairment, was evaluated by open field test, elevated plus maze, and fear conditioning. Thereafter, pathological changes of neurodegeneration were explored by western blot and immunofluorescence assays.

Results: Selegiline administration significantly ameliorated TF-induced impulsive-like behaviors and reduced excessive GABA production in reactive hippocampal astrocytes. Moreover, astrocyte-specific NLRP3 knockout mice reversed TF-induced impulsive-like and cognitive impairment behaviors, decreased GABA levels in reactive astrocytes, ameliorated NLRP3-associated inflammatory responses during the early stage, and restored neuronal degeneration in the hippocampus.

Conclusions: Our findings suggest that anesthesia and surgical procedures trigger neuroinflammation and cognitive deficits, which may be due to NLRP3-GABA activation in the hippocampus of aged mice.

Keywords: Astrocyte; GABA; NLRP3; Perioperative neurocognitive disorders; Selegiline.

Fig. 1

Experimental schematic diagram. Perioperative neurocognitive disorders (PND) were established by tibial fracture surgery (TF) in aged mice. At 8 days after TF exposure, open field test (OFT), elevated plus maze (EPM), and the first stage of fear conditioning (FC) were performed. At 9 days after TF exposure, the second stage of FC was performed. At 10 days after TF exposure, the immunofluorescence (IF) and western blot assay (WB) experiments were performed. Selegiline was administered from postoperative day 0 to 7 once a day. An equal volume of normal saline was used as vehicle

Fig. 2

Anesthesia and surgery caused impulsive-like behaviors and cognitive deficits in aged mice. A Computer printouts showing the shifting trajectories of each group in the OFT at 8 days after surgical exposure. B The total distance for each group at 8 days after surgical exposure. C Computer printouts showing the shifting trajectories of each group in the EPM at 8 days after surgical exposure. D The time spent in the open arm for each group at 8 days after surgical exposure. E FC test experimental schematic diagram. F Freezing time during the FC test results caused by the indicated stimuli. Data are presented as the mean ± SD (n = 12 mice/group). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test or Kruskal–Wallis and Dunn’s multiple comparison test. **P < 0.01

Fig. 3

Anesthesia and surgery lead to neuronal damage, reactive astrocytes, and increased excitatory neurons. A Representative photomicrographs of GFAP-, NeuN-, VGLUT2-, GAD65-, GABA- and glutamate-positive cells in the CA1 of the hippocampus. Scale bar = 50 μm. B–E The intensity of GFAP, NeuN, VGLUT2 and GAD65 was quantified in each group. F, G Co-stained area of GABA- and GFAP-positive cells, and glutamate- and GFAP-positive cells in the CA1 of the hippocampus. H Representative western blot of MAP2 and PSD95. I, J The optical density values of MAP2 and PSD95 in the hippocampus. Data are presented as the mean ± SD (n = 6 mice/group). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test or Kruskal–Wallis and Dunn’s multiple comparison test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05

Fig. 4

Selegiline administration ameliorates impulsive-like behaviors but not cognitive impairment post-surgery. A Computer printouts showing the shifting trajectories of each group in the OFT at 8 days after surgical exposure. B Computer printouts showing the shifting trajectories of each group in the EPM at 8 days after surgical exposure. C The total distance for each group at 8 days after surgical exposure. D The time spent in the open arm for each group at 8 days after surgical exposure. E The freezing time caused by the indicated stimuli during the FC test results. Data are presented as the mean ± SD (n = 12 mice/group). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test or Kruskal–Wallis and Dunn’s multiple comparison test. ****P < 0.0001; *P < 0.05

Fig. 5

The imbalance between excitatory and inhibitory neurons can be restored by selegiline administration. A Representative photomicrographs of GFAP-, NeuN-, VGLUT2-, GAD65-, GABA- and glutamate-positive cells in the CA1 of hippocampus at 10 days after surgical exposure. Scale bar = 50 μm. B Image of CA1 in hippocampus. C–F The intensity of GFAP, NeuN, VGLUT2, and GAD65 was quantified in each group. G, H Co-stained area of GABA- and GFAP-positive cells, and glutamate- and GFAP-positive cells in the CA1 of hippocampus. I Representative western blot of IL-1β, IL-18, MAP2, and PSD95. J–M The optical density values of IL-1β, IL-18, MAP2, and PSD95. Data are presented as the mean ± SD (n = 6 mice/group). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test or Kruskal–Wallis and Dunn’s multiple comparison test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05

Fig. 6

Cognitive impairment may be related to pyroptosis-associated inflammatory responses in astrocytes. A Representative photomicrographs of NLRP3-, IL-18-, IL-1β- and cleaved caspase-1-positive cells in the CA1 of the hippocampus at 12 h and 10 days after surgical exposure. Scale bar = 50 μm. B Image of CA1 in hippocampus. C–J Co-stained area of NLRP3-, IL-18-, IL-1β-, cleaved caspase-1- and GFAP-positive cells in the CA1 of the hippocampus. K Representative western blot of NLRP3, IL-1β, IL-18 and cleaved caspase-1 at 12 h after surgical exposure. L–O The optical density values of NLRP3, IL-1β, IL-18, and cleaved caspase-1 at 12 h after surgical exposure. P Representative western blot of NLRP3, IL-1β, IL-18 and cleaved caspase-1 at 10 days after surgical exposure. Q–T The optical density values of NLRP3, IL-1β, IL-18 and cleaved caspase-1 at 10 days after surgical exposure. Data are presented as the mean ± SD (n = 6 mice/group or n = 3 mice/group). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test or Kruskal–Wallis and Dunn’s multiple comparison test. ***P < 0.001; **P < 0.01; *P < 0.05

Fig. 7

Astrocyte-specific NLRP3 knockout ameliorates impulsive-like behaviors and cognitive impairment post-surgery. A Breeding scheme for GFAP-Cre mice crossed with NLRP3-cKO mice and gene strategy for preparing NLRP3-conditional knockout (cKO) mice. B Representative genotyping analysis for NLRP3-cKO homozygote, NLRP3-cKO heterozygote, and WT (wild type) mice and NLRP3-cKO homozygote, NLRP3-cKO heterozygote, GFAP-Cre, non-GFAP-Cre F4 mice. C Computer printouts showing the shifting trajectories of each group in the OFT at 8 days after surgical exposure. D Computer printouts showing the shifting trajectories of each group in the EPM at 8 days after surgical exposure. E The total distance for each group at 8 days after surgical exposure. F The time spent in the open arm for each group at 8 days after surgical exposure. G The freezing time caused by the indicated stimuli during the FC test results. Data are presented as the mean ± SD (n = 12 mice/group). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test or Kruskal–Wallis and Dunn’s multiple comparison test. ****P < 0.0001; *P < 0.05

Fig. 8

Astrocyte-specific NLRP3 knockout mitigated reactive astrocytes and excessive GABA, and improved neuronal degeneration. A Image of CA1 in hippocampus. Representative photomicrographs of GFAP-, NeuN-, VGLUT2-, GAD65-, GABA-, glutamate- and NLRP3-positive cells in the CA1 of hippocampus at 10 days after surgery. Scale bar = 50 μm. B–E The intensity of GFAP, NeuN, VGLUT2 and GAD65 was quantified in each group. F–H Co-stained area of GABA-, glutamate-, and NLRP3- and GFAP-positive cells in the CA1 of the hippocampus. I Representative western blot of IL-1β, IL-18, MAP2, PSD95 and cleaved caspase-1 at 10 days after surgery. J–N The optical density values of IL-1β, IL-18, MAP2, PSD95, and cleaved caspase-1. O Representative photomicrographs of IL-1β-, IL-18- and cleaved caspase-1-positive cells in the CA1 of the hippocampus at 12 h after surgical exposure. Scale bar = 50 μm. P–R Co-stained area of IL-1β-, IL-18-, cleaved caspase-1- and GFAP-positive cells in the CA1 of the hippocampus. Data are presented as the mean ± SD (n = 6 mice/group or n = 3 mice/group). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test or Kruskal–Wallis and Dunn’s multiple comparison test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05