Gut microbiota-derived metabolite trimethylamine N-oxide aggravates cognitive dysfunction induced by femoral fracture operation in mice

Abstract

An increasing number of elderly individuals are experiencing postoperative cognitive dysfunction (POCD) problems after undergoing hip replacement surgery, with gut microbiota metabolites playing a role in its pathogenesis. Among these, the specific effects of trimethylamine N-oxide (TMAO) on POCD are still unclear. This study aimed to explore the role of TMAO on cognitive dysfunction and underlying mechanisms in mice. The POCD model was created through femoral fracture surgery in elderly mice, followed by cognitive function assessments using the Morris Water Maze and Novel Object Recognition tests. The gut microbiota depletion and fecal microbiota transplantation were performed to examine the relationship between TMAO levels and cognitive outcomes. The effects of TMAO treatment on cognitive dysfunction, microglial activation, and inflammatory cytokine levels in the brain were also evaluated, with additional assessment of the role of microglial ablation in reducing TMAO-induced cognitive impairment. Elevated TMAO levels were found to be associated with cognitive decline in mice following femoral fracture surgery, with gut microbiota depletion mitigating both TMAO elevation and cognitive dysfunction. In contrast, fecal microbiota transplantation from postoperative mice resulted in accelerated cognitive dysfunction and TMAO accumulation in germ-free mice. Furthermore, TMAO treatment worsened cognitive deficits, neuroinflammation, and promoted microglial activation, which were reversed through the ablation of microglia. TMAO exacerbates cognitive dysfunction and neuroinflammation in POCD mice, with microglial activation playing a crucial role in this process. Our findings may provide new therapeutic strategies for managing TMAO-related POCD and improving the quality of life for elderly patients.

Keywords: inflammatory cytokine; postoperative cognitive dysfunction; quality of life; trimethylamine N‐oxide; underlying mechanisms.

FIGURE 1

Elevated cerebral TMAO levels was observed and associated with cognitive dysfunction in femoral fracture operation mouse model. (A) A schematic representation of the experimental timeline, showing the establishment of the femoral fracture model, followed by water maze navigation training from the 3rd to the 7th‐day post‐operation, and concluding with a space exploration test on the 8th day. (B) The mean latency, (C) number of platform crossings, and time spent in the target quadrant of mice in the Morris water maze test. (D) The novel object recognition was subsequently performed and the exploration index of new objects was calculated to determine recognition memory deficits. (E) The levels of TMAO in the (E) plasma, (F) brain, and (G) intestines of POCD mice were assessed through high‐performance liquid chromatography‐mass spectrometry and the correlation between intestinal TMAO concentrations with (H) escape latency, (I) the number of crossings over the target zone and (J) the exploration index for novel objects were examined by the Pearson's correlation test. Data was represented as mean ± SEM. ***p < 0.001.

FIGURE 2

Impact of gut microbiota depletion on TMAO levels and cognitive dysfunction in a femoral fracture operation mouse model. (A) Schematic representation of the experimental setup, showing a week‐long antibiotic (ABX) pretreatment before femoral fracture surgery to evaluate effects on TMAO concentrations, cognitive performance, and neuroinflammation. (B) Comparison of body weights between ABX‐treated and PBS‐treated control mice. (C) TMAO concentrations in brain, intestines, and plasma following femoral fracture operation. (D) Novel object recognition test and (E, F) Morris water maze test were performed to assess cognitive function affected by gut microbiota depletion. (G) ELISA analysis was employed to detect inflammatory markers in brain tissues. Data represented as mean ± SEM, with **p < 0.01, ***p < 0.001 between the Sham and POCD group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 between the POCD and ABX + POCD group; ns, no significance.

FIGURE 3

Acceleration of cognitive dysfunction and TMAO accumulation in germ‐free mice through fecal microbiota transplantation (FMT) from femoral fracture‐operated mice. (A) A schematic illustration of the FMT process from donors (normal or femoral fracture operation‐treated mice) to germ‐free recipients. Following FMT, the cognitive function of mice was evaluated through (B) escape latency, (C) the number of platform crossings, time spent in the target quadrant, and (D) the exploration index utilizing the Morris water maze test or novel object recognition test. The TMAO levels in (E) plasma, (F) brain, and (G) intestines were identified to determine the effect of FMT on TMAO accumulation. (H–K) Proinflammatory cytokines demonstrate the inflammatory profiling of brain tissues after FMT from femoral fracture operation‐treated mice. Data are presented as mean ± SEM. Statistical significance is denoted by ***p < 0.001.

FIGURE 4

Effects of TMAO treatment on cognitive dysfunction and microglial activation in a femoral fracture operation mouse model. (A) Experimental design showing oral administration of TMAO at a dose of 180 mg/kg for 3 weeks before femoral fracture surgery. (B, C) Morris water maze test and (D) the novel object recognition test revealed diminished exploration indexes in TMAO‐treated groups. (E) Increased levels of inflammatory cytokines IL‐1β, IL‐6, TNF‐α, and IL‐10 in brain tissues indicate increased neuroinflammation due to TMAO exposure. (F) Immunohistochemistry for Iba‐1 was carried out to detect microglial activation, characterized by denser brown precipitates. (G) Flow cytometry analysis on CD86 and CD206 positive cells was conferred to reveal microglial polarization. Data are demonstrated as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Sham vs. Sham + TMAO); ^### p < 0.001 (Sham vs. POCD); ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001 (POCD vs. POCD + TMAO); ns, no significance.

FIGURE 5

Microglia activation was essential for TMAO‐mediated cognitive dysfunction during femoral fracture operation. (A) Diagram illustrating PLX5622 administration for one week before TMAO treatment to inactivate microglia. (B) Immunohistochemistry for Iba‐1 in brain sections. (C) Escape latency, (D) frequency of platform crossings and duration spent in the target platform location were obtained from the Morris water maze test. (E) NOR test was utilized to show exploration indexes of mice for new objects. (F) The inflammatory cytokine levels in brain tissues were detected to determine neuroinflammation via ELISA. Data was represented as mean ± SEM. ***p < 0.001 (POCD vs. POCD + PLX5622); ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 (POCD vs. POCD + TMAO); ^&&& p < 0.001 (POCD + TMAO vs. POCD + PLX5622 + TMAO); ns, no significance.