Methylene Blue Cuts Postoperative Delirium by Calming Brain Inflammation

Postoperative delirium (POD) is an acute brain dysfunction syndrome affecting patients across all age groups after surgery and anesthesia, with the elderly at highest risk. It is linked to prolonged hospitalization, increased healthcare costs, functional decline, and higher mortality. Despite its clinical burden, no proven pharmacological treatments exist, partly because the underlying biology remains incompletely understood. This study investigates whether methylene blue (MB), an inexpensive FDA-approved compound with known anti-inflammatory and neuroprotective properties, can reduce POD-like behavior in mice.

Adult mice underwent laparotomy under 1.4% isoflurane anesthesia. Four groups were studied: control, MB-only, anesthesia/surgery (A/S), and A/S plus MB (5 mg/kg intraperitoneally). A battery of behavioral tests — buried food test, open-field test, and Y-maze — was conducted 24 hours pre-surgery and at 6 and 24 hours post-surgery. Composite Z-scores integrating all behavioral measures were calculated to quantify delirium-like severity.

POD-like behavior was most pronounced at 6 hours post-surgery, with the A/S group showing significantly worse food-finding latency, reduced time in the open-field center (reflecting anxiety), and fewer entries and less time in the novel arm of the Y-maze (reflecting spatial memory impairment). MB treatment normalized these deficits. By 24 hours, behavioral differences between groups largely disappeared, consistent with the transient, fluctuating nature of clinical delirium.

Neuroinflammation analysis at 6 hours post-surgery revealed significantly elevated mRNA levels of TNF-α, IL-6, IL-1β, complement components C1q and C3, and NLRP3 inflammasome in both the hippocampus and prefrontal cortex of A/S mice. MB suppressed all of these. Microglial activation, assessed by Iba-1 immunoblotting and immunofluorescence, showed amoeba-like (activated) morphology in A/S mice that MB reversed toward ramified (resting) shapes. Reactive oxygen species (ROS), measured by DHE staining in the prefrontal cortex, were also significantly elevated after A/S and reduced by MB.

Blood-brain barrier integrity was assessed through IgG immunofluorescence (a marker of vascular leakage), 10-kDa dextran permeability, tight junction protein expression (claudin-5, occludin, ZO-1), and transcytosis markers. A/S caused significant BBB leakage in both hippocampus and prefrontal cortex. MB restored tight junction protein levels, reduced EMMPRIN and MMP9 (proteins that degrade the BBB matrix), and decreased transcytosis. Together, these results demonstrate that MB repairs BBB integrity through multiple complementary mechanisms. The authors conclude that MB's dual action — suppressing neuroinflammation and restoring BBB function — underlies its protection against POD-like behavior in this preclinical model.