Critical role of NLRP3-caspase-1 pathway in age-dependent isoflurane-induced microglial inflammatory response and cognitive impairment

Abstract

Background: Elderly patients are more likely to suffer from postoperative cognitive dysfunction (POCD) after surgery and anesthesia. Except for declined organ function, the particular pathogenesis of POCD in elderly patients remains unknown. This study is carried out to determine the critical role of the NOD-like receptor protein 3 (NLRP3)-caspase-1 pathway in isoflurane-induced cognitive impairment.

Methods: Young (6-8 months old) and aged (14 months old) healthy male C57BL/6 mice were exposed to 1.5% isoflurane for 2 h. Some mice received intraperitoneal injection of Ac-YVAD-cmk (8 mg/kg), a specific inhibitor of caspase-1, 30 min before the isoflurane exposure. Morris water maze test was carried out 1 week after the isoflurane anesthesia. Brain tissues were harvested 24 h after the isoflurane anesthesia. Western blotting was carried out to detect the expression of NLRP3, interleukin (IL)-1β, and IL-18 in the hippocampus. Mouse microglial cell line BV-2 and primary microglial cultures were primed by lipopolysaccharide for 30 min before being exposed to isoflurane. NLRP3 was downregulated by RNA interference.

Results: Compared to young mice, aged mice had an increased expression of NLRP3 in the hippocampus. Isoflurane induced cognitive impairment and hippocampal inflammation in aged mice but not in young mice. These effects were attenuated by Ac-YVAD-cmk pretreatment (P < 0.05). Isoflurane activated NLRP3-caspase-1 pathway and increased the secretion of IL-18 and IL-1β in cells pretreated with lipopolysaccharide but not in cells without pretreatment. Downregulation of NLRP3 attenuated the activation of NLRP3 inflammasome by isoflurane.

Conclusions: NLRP3 priming status in aged mouse brain may be involved in isoflurane-induced hippocampal inflammation and cognitive impairment.

Keywords: Aging; Isoflurane; NOD-like receptor protein 3 inflammasome; Neuroinflammation; Postoperative cognitive dysfunction.

Fig. 1

Isoflurane induced age-related cognitive impairment. Young (6–8 months old) and aged (14 monts old) healthy male C57BL/6 mice were exposed to 1.5% isoflurane for 2 h. Some aged mice received intraperitoneal injection of 8 mg/kg Ac-YVAD-cmk 30 min before the isoflurane exposure. YC = blank control of young mice; Y-ISO = young mice exposed to isoflurane; AC = blank control of aged mice; A-ISO = aged mice exposed to isoflurane; A-ISO-cmk = Ac-YVAD-cmk administered before aged mice exposed to isoflurane; A-ISO-PBS = solvent of Ac-YVAD-cmk (PBS+ less than 1% DMSO) administered before aged mice exposed to isoflurane. Morris water maze test was carried out 1 week after the isoflurane anesthesia. a, b, and c Escape latency to reach the platform. d Target quadrant traveling time. Normalization of the target quadrant traveling time of group YC and Y-ISO was by the mean of group YC, and group AC, A-ISO, A-ISO-cmk, and A-ISO-PBS by the mean of group AC. e Platform-crossing times. f Average swimming speed on the sixth day. All results are mean ± SD (n = 15). #P < 0.05 and ##P < 0.01 compared with the corresponding data of group AC. *P < 0.05 compared with the corresponding data of group A-ISO

Fig. 2

Contribution of NLRP3 inflammasome activation to isoflurane-induced age-related neuroinflammation. Hippocampus was harvested at 24 h after the anesthesia. a Representative western blot images of NLRP3, caspase-1 P45, caspase-1 P20, ASC, IL-18, IL-1β, cleaved caspase-3, and Iba-1. b–i Graphic presentation of abundance of each protein. All values are expressed as fold changes over the mean values of group YC and are presented as mean ± SD (n ≥ 3). *P < 0.05 compared with the corresponding data of group YC. #P < 0.05 and ##P < 0.05 compared with the corresponding data of group AC

Fig. 3

NLRP3 priming was induced by LPS stimulation. Different doses of LPS were added to the cell culture media of BV-2 cells or primary microglial cultures for 30 min. a The mRNA of NLRP3 was quantified by real-time qPCR immediately after LPS stimulation. Values are expressed as fold changes over the mean values of blank control. b IL-1β concentration in the supernatant was measured 6 h later. All results are presented as mean ± SD (n ≥ 3). *P < 0.05 and **P < 0.01 compared with the corresponding data of blank control

Fig. 4

Isoflurane-induced NLRP3 inflammasome activation after priming. BV-2 microglia were exposed to 4% isoflurane for 6 h in the cells primed with or without 1 μg/mL LPS. Primary microglial cultures were exposed to 2% isoflurane for 6 h in the cells primed with or without 5 ng/mL LPS. Control = blank control; ISO = Isoflurane exposure; NLRP3-primed = LPS stimulation; NLRP3-primed+ ISO = NLRP3-primed + isoflurane exposure. a Western blot images of NLRP3, caspase-1 P45, caspase-1 P20, ASC, IL-18, and IL-1β from BV-2 cells. b–g Graphic presentation of abundance of each protein in BV-2 cells. h Western blot images of NLRP3, ASC, caspase-1 P45, and caspase-1 P20, IL-18, and IL-1β. i–n Graphic presentation of abundance of each protein in primary microglia. Values are expressed as fold changes over the mean values of control and are presented as mean ± SD (n ≥ 3). *P < 0.05 and **P < 0.01 compared with the corresponding data of group control. #P < 0.05 and ##P < 0.01 compared with the corresponding data of group NLRP3-primed

Fig. 5

NLRP3 priming was necessary in isoflurane-induced IL-1β production. BV-2 cells primed with or without 1 μg/mL LPS were exposed to 4% isoflurane for 6 h. Primary microglial cultures primed with or without 5 ng/mL LPS were exposed to 2% isoflurane for 6 h. Control = blank control; ISO = isoflurane exposure; NLRP3-primed = LPS stimulation; NLRP3-primed + ISO = NLRP3-primed + isoflurane exposure. a The mRNA of NLRP3 in BV-2 cells. Values are expressed as fold changes over the mean values of blank control and are presented as mean ± SD (n = 6). b IL-1β concentration in the supernatant of BV-2 cells. c Viability of NLRP3-primed cells at 0 and 12 h after isoflurane exposure. Values are expressed as fold changes over the mean values of NLRP3-primed cells and are presented as mean ± SD (n = 3). d The mRNA of NLRP3 in primary microglial cultures. Values are expressed as fold changes over the mean values of blank control and are presented as mean ± SD (n = 3). e IL-1β concentration in the supernatant of primary microglial cultures. Values are expressed as fold changes over the mean values of control and are presented as mean ± SD (n = 3). *P < 0.05 and **P < 0.01 compared with the corresponding data of group control. #P < 0.05 and ##P < 0.01 compared with the corresponding data of group NLRP3-primed cells

Fig. 6

NLRP3 knock-down reduced isoflurane-induced NLRP3 inflammasome activation. Cells were transfected with NLRP3 siRNA or negative control siRNA 24 h before NLRP3 priming and exposure to 4% isoflurane for 6 h. NC-siRNA + ISO = negative control of siRNA before NLRP3 priming and isoflurane exposure; NLRP3-siRNA + ISO = NLRP3 siRNA before NLRP3 priming and isoflurane exposure. a Western blot images of NLRP3, caspase-1 P45, caspase-1 P20, ASC, IL-18, and IL-1β. b–g The graphic presentation of each protein abundance of Fig. 6a. h Whole cell protein samples from BV-2 cells were harvested immediately after isoflurane treatment. Immunoprecipitation was performed by using an anti-NLRP3 antibody and was immunoblotted for NLRP3, ASC, and pro-caspase-1. Values are expressed as fold changes over the mean values of NLRP3-primed cells and are presented as mean ± SD (n = 6). *P < 0.05 compared with the corresponding data of group control. #P < 0.05 compared with the corresponding data of NLRP3-primed + ISO