Dysregulation of BDNF/TrkB signaling mediated by NMDAR/Ca2+/calpain might contribute to postoperative cognitive dysfunction in aging mice

Abstract

Background: Postoperative cognitive decline (POCD) is a recognized clinical phenomenon characterized by cognitive impairments in patients following anesthesia and surgery, yet its underlying mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity, learning, and memory via activation of TrkB-full length (TrkB-FL) receptors. It has been reported that an abnormal truncation of TrkB mediated by calpain results in dysregulation of BDNF/TrkB signaling and is associated with cognitive impairments in several neurodegenerative disorders. Calpains are Ca²⁺-dependent proteases, and overactivation of calpain is linked to neuronal death. Since one source of intracellular Ca²⁺ is N-methyl-d-aspartate receptors (NMDARs) related and the function of NMDARs can be regulated by neuroinflammation, we therefore hypothesized that dysregulation of BDNF/TrkB signaling mediated by NMDAR/Ca²⁺/calpain might be involved in the pathogenesis of POCD.

Methods: In the present study, 16-month-old C57BL/6 mice were subjected to exploratory laparotomy with isoflurane anesthesia to establish the POCD animal model. For the interventional study, mice were treated with either NMDAR antagonist memantine or calpain inhibitor MDL-28170. Behavioral tests were performed by open field, Y maze, and fear conditioning tests from 5 to 8 days post-surgery. The levels of Iba-1, GFAP, interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), NMDARs, calpain, BDNF, TrkB, bax, bcl-2, caspase-3, and dendritic spine density were determined in the hippocampus.

Results: Anesthesia and surgery-induced neuroinflammation overactivated NMDARs and then triggered overactivation of calpain, which subsequently led to the truncation of TrkB-FL, BDNF/TrkB signaling dysregulation, dendritic spine loss, and cell apoptosis, contributing to cognitive impairments in aging mice. These abnormities were prevented by memantine or MDL-28170 treatment.

Conclusion: Collectively, our study supports the notion that NMDAR/Ca2+/calpain is mechanistically involved in anesthesia and surgery-induced BDNF/TrkB signaling disruption and cognitive impairments in aging mice, which provides one possible therapeutic target for POCD.

Keywords: BDNF; Calpain; Cognitive dysfunction; NMDAR; Neuroinflammatioin; Surgery; TrkB.

Fig. 1

Schematic timeline of the experimental procedure. The mice were treated with MEM (a NMDAR antagonist, 20 mg/kg) or MDL-28170 (a calpain inhibitor, 20 mg/kg) intraperitoneally (i.p.) before anesthesia, and the surgery was performed immediately after 30 min exposure to isoflurane. Twenty-four hours later, the mice were sacrificed for Western blot and immunofluorescence detection. For the behavioral study, mice underwent anesthesia and surgery on day 0. The mice were treated with MEM (20 mg/kg) or MDL-28170 (20 mg/kg) i.p. before anesthesia, and once daily for the subsequent 5 consecutive days. Behavioral tests were performed from 5 to 8 days post-surgery with open field, Y maze, and fear conditioning tests, respectively. Three hours after behavioral tests at 8 days post-surgery, the brains of mice were collected for Western blot and Golgi-Cox staining

Fig. 2

Anesthesia and surgery-induced activation of microglia and astrocytes in the hippocampus was attenuated by MEM treatment on day 1 post-surgery. a Representative images of Iba-1 (a marker of microglia) in the hippocampus. b Representative images of GFAP (a marker of astrocytes) in the hippocampus. c Quantification of Iba-1 fluorescence. d Quantification of GFAP fluorescence. Data are presented as the mean ± SEM (n = 6). *p < 0.05 compared to the con + veh group (**p < 0.01, ***p < 0.001), ^#p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001). DAPI staining is shown in blue. Scale bar = 50 μm

Fig. 3

Increased hippocampal levels of IL-1β, IL-6, and NMDAR subunits after anesthesia and surgery were attenuated by MEM treatment on day 1 post-surgery. a Representative Western blots of IL-1β in the hippocampus. GAPDH was included as a loading control. b Quantitative analysis of IL-1β levels. c Representative Western blots of IL-6 in the hippocampus. GAPDH was included as loading control. d Quantitative analysis of IL-6 levels. e Representative Western blots of TNF-α in the hippocampus. GAPDH was included as loading control. f Quantitative analysis of TNF-α levels. g Representative Western blots of GluN2A. GAPDH was included as loading control. h Quantitative analysis of GluN2A levels. i Representative Western blots of GluN2B. GAPDH was included as loading control. j Quantitative analysis of GluN2B levels. Data are presented as the mean ± SEM (n = 6). *p < 0.05 compared to the con + veh group (**p < 0.01, ***p < 0.001), ^#p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001)

Fig. 4

Increased hippocampal levels of IL-1β and IL-6 after anesthesia and surgery were detected on day 8 post-surgery, which were attenuated by MEM treatment. a Representative Western blots of IL-1β in the hippocampus. β-tubulin was included as loading control. b Quantitative analysis of IL-1β levels. c Representative Western blots of IL-6 in the hippocampus. β-tubulin was included as loading control. d Quantitative analysis of IL-6 levels. Data are presented as the mean ± SEM (n = 6). *p < 0.05 compared to the con + veh group (**p < 0.01, ***p < 0.001), #p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001)

Fig. 5

MEM treatment reduced the cleavage of TrkB by modulating the overactivation of calpain on day 1 post-surgery. a Representative Western blots of SBDP in the hippocampus. GAPDH was included as loading control. b Quantitative analysis of the ratio of the SBDP to αII-spectrin. c Representative Western blots of TrkB-FL. GAPDH was included as loading control. d Quantitative analysis of TrkB-FL levels. e Representative Western blots of TrkB-ICD. β-tubulin was included as loading control. f Quantitative analysis of TrkB-ICD levels. Data are presented as the mean ± SEM (n = 6). *p < 0.05 compared to the con + veh group (**p < 0.01, ***p < 0.001), #p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001)

Fig. 6

Inhibition of NMDAR or calpain restored BDNF/TrkB signaling disruption on day 1 post-surgery. a Representative Western blots of SBDP in the hippocampus. β-tubulin was included as loading control. b Quantitative analysis of the ratio of the SBDP to αII-spectrin. c Representative Western blots of BDNF in the hippocampus. β-tubulin was included as loading control. d Quantitative analysis of BDNF levels. e Representative Western blots of TrkB-FL. β-tubulin was included as loading control. f Quantitative analysis of TrkB-FL levels. g Representative Western blots of TrkB-ICD. β-tubulin was included as loading control. h Quantitative analysis of TrkB-ICD levels. Data are presented as the mean ± SEM (n = 6). *p < 0.05 compared to the con + veh group (**p < 0.01, ***p < 0.001), ^#p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001)

Fig. 7

Increased levels of markers associated with cell apoptosis were attenuated by MEM or MDL-28170 treatment on day 1 post-surgery. a Representative Western blots of bax in the hippocampus. β-tubulin was included as loading control. b Quantitative analysis of bax. c Representative Western blots of bcl-2. β-tubulin was included as loading control. d Quantitative analysis of bcl-2 levels. e Representative Western blots of FL-caspase-3 and cleaved caspase-3 in the hippocampus. β-tubulin was included as loading control. f Quantitative analysis of cleaved caspase-3 and FL-caspase-3 levels. g Representative western-blots of GluN2A. β-tubulin was included as loading control. h Quantitative analysis of GluN2A levels. i Representative Western blots of GluN2B. β-tubulin was included as loading control. j Quantitative analysis of GluN2B levels. Data are presented as the mean ± SEM (n = 6). *p < 0.05 compared to the con+ veh group (**p < 0.01, ***P < 0.001), #p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001)

Fig. 8

Inhibition of NMDAR or calpain attenuated hippocampal dendritic density loss after anesthesia and surgery. a A hippocampal profile image of Golgi-Cox staining. b Representative camera tracings of hippocampal CA1 neurons of the six groups. c Quantitation of the dendritic intersections of the six groups. d Quantitation of the total dendritic length of the six groups. e Representative dendritic spine density of hippocampal CA1 neurons of the six groups. f Quantitation of the dendritic spine density of the six groups. Data are presented as the mean ± SEM (n = 6). *p < 0.05 compared to the con + veh group (**p < 0.01, ***p < 0.001), ^#p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001)

Fig. 9

Anesthesia and surgery-induced cognitive impairments were attenuated by MEM or MDL-28170 treatment. a, b There was no significant difference at ambulatory distance and time spent in the center in the open field test among the six groups at 5 days post-surgery. c There was no significant difference of the total arm entries in the Y maze test. d The mice in the sur + veh group displayed lesser spontaneous alteration than the mice in the con + veh group, which was reversed by MEM or MDL-28170 treatment. e The freezing time to context was significantly decreased after anesthesia and surgery, while MEM or MDL-28170 treatment evidently increased the freezing time. Data are presented as the mean ± SEM (n = 10). *p < 0.05 compared to the con+ veh group (**p < 0.01, ***p < 0.001), ^#p < 0.05 compared to the sur + veh group (^##p < 0.01, ^###p < 0.001)

Fig. 10

General overview of the main highlights of this study. Anesthesia and surgery-induced neuroinflammation overactivated NMDARs and calpain, which subsequently led to the truncation of TrkB-FL, BDNF/TrkB signaling dysregulation, dendritic spine loss, and cell apoptosis, contributing to cognitive impairments in aging mice