Microglia ameliorate delirium-like phenotypes in a murine model of acute ventilator-induced lung injury

Abstract

Background: Delirium affects 50-85% of patients on mechanical ventilation and is associated with increased mortality, prolonged hospitalization, and a three-fold higher risk of dementia. Microglia, the resident immune cells of the brain, exhibit both neuroprotective and neurotoxic functions; however, their effects in mechanical ventilation-induced acute lung injury (VILI) are unknown. We hypothesize that in a model of short-term VILI, microglia play a neuroprotective role to ameliorate delirium-like phenotypes.

Methods: Microglia depletion (n = 18) was accomplished using an orally administered colony stimulating factor 1 receptor inhibitor, while controls received a vehicle diet (n = 18). We then compared extent of neuronal injury in the frontal cortex and hippocampus using cleaved caspase-3 (CC3) and multiple delirium-like behaviors in microglia depleted and non-microglia depleted male mice (C57BL/6 J aged 4-9 months) following VILI. Delirium-like behaviors were evaluated using the Open Field, Elevated Plus Maze, and Y-maze assays. We subsequently evaluated whether repopulation of microglia (n = 14 repopulation, 14 vehicle) restored the phenotypes.

Results: Frontal/hippocampal neuronal CC3 levels were significantly higher in microglia depleted VILI mice compared to vehicle-treated VILI controls (p < 0.01, p < 0.01, respectively). These structural changes were accompanied by worse delirium-like behaviors in microglia depleted VILI mice compared to vehicle controls. Specifically, microglia depleted VILI mice demonstrated: (1) significantly increased time in the periphery of the Open Field (p = 0.01), (2) significantly increased coefficient of variation (p = 0.02), (3) trend towards reduced time in the open arms of the Elevated Plus Maze (p = 0.09), and (4) significantly decreased spontaneous alternations on Y-maze (p < 0.01). There was a significant inverse correlation between frontal CC3 and percent spontaneous alternations (R² = 0.51, p < 0.01). Microglia repopulation showed a near-complete return to vehicle levels of delirium like-behaviors.

Conclusions: This study demonstrates that microglia depletion exacerbates structural and functional delirium-like phenotypes after VILI, while subsequent repopulation of microglia restores these phenotypes. These findings suggest a neuroprotective role for microglia in ameliorating neuronal and functional delirium-like phenotypes and call for consideration of interventions that leverage endogenous microglia physiology to mitigate delirium.

Keywords: Delirium; Mechanical ventilation; Microglia; Ventilator-induced lung injury.

Fig. 1

A Timeline of microglia depletion study. 13 mice were treated with 600 mg/kg Pexidartinib (PEXI) in chow and 12 were given a vehicle-matched diet for two weeks before conducting behavioral tests and inducing VILI, after which they recovered for 15 h before another round of behavioral testing was done and tissue was collected. B Oxygen (O₂) saturation during mechanical ventilation was not significantly different between treatment groups in the first experiment (1A). C Pulmonary inflammation, measured by percentage of polymorphonuclear cells (PMNs) in the bronchoalveolar lavage fluid (BALF), was not significantly different between treatment groups post-VILI (n = 10 PEXI, n = 12 vehicle, t = 1.12, p = 0.28). Two mice from the PEXI group died during ventilation and BALF could not be obtained from one PEXI mouse. D Plasma IL-6 levels were not significantly different between treatment groups post-VILI (n = 7 PEXI, n = 11 vehicle, t = 0.34, p = 0.74). Residuals were observed and three statistical outliers (ROUT Q = 1%) were removed from plasma IL-6 analysis (2 vehicle, 1 PEXI)

Fig. 2

Microglia depletion exacerbates VILI-induced neuronal injury. A, B Frontal and hippocampal neuronal CC3 were significantly higher in PEXI- compared to vehicle-treated VILI mice (Welch’s t test, n = 11/group, t = 4.814, p < 0.01 and n = 11 PEXI and 10 vehicle, t = 9.42, p < 0.01, respectively). C, D Frontal and hippocampal Iba-1 were significantly decreased in PEXI-compared to vehicle-treated VILI mice (Welch’s t test, n = 17, t = 18.85, p < 0.01 and n = 17, t = 8.851, p < 0.01, respectively). E Representative images of neuronal CC3 in vehicle and PEXI-treated mice, respectively. F Representative Iba-1 fluorescence microscopy images of vehicle and PEXI-treated mice, respectively. G 20 × representative images of CC3 and IL-6 in vehicle and PEXI-treated mice, respectively. H After mechanical ventilation, PEXI-treated mice had significantly higher frontal IL-6 (Welch’s t test, n = 22, t = 3.288, p = 0.01). One vehicle VILI animal had no usable tissue for any stain. Two PEXI VILI animals died during ventilation and had no usable tissue. Other animals were excluded from histological analyses if less than two brain sections were quantifiable for the specific stain (CC3 Hippocampus: 1 vehicle VILI; Iba-1 Frontal: 1 PEXI VILI, 4 vehicle VILI; Iba-1 Hippocampus: 1 PEXI VILI, 4 vehicle VILI). Quantitative data are expressed in mean ± SEM. Residuals were observed and there were no influential outliers

Fig. 3

Microglia depletion exacerbates VILI-induced delirium-like behaviors. A Time spent in the periphery of the Open Field arena was significantly higher in PEXI- compared to vehicle-treated VILI mice (n = 8 PEXI, n = 10 vehicle, t = 2.559, p = 0.01), indicating increased anxiety-related behavior. B The mean visit duration to the periphery zone was significantly higher in PEXI- compared to vehicle-treated VILI mice (n = 7 PEXI, n = 8 vehicle, t = 4.161, p < 0.001) C, D Representative heat maps of mouse movement during the 45 min Open Field test show PEXI-treated mice significantly increased time spent in the periphery after injury. This is indicated by the appearance of more red coloring near the walls of the apparatus, which signifies areas where the mice spent more time over the test duration. Two PEXI VILI animals died during ventilation and thus have no post VILI behavior. Animals were excluded from analyses if they did not participate in a specific behavioral assay, exclusion criteria are defined in the Methods section (Open Field: post VILI—2 vehicle, 2 PEXI). Quantitative data are expressed in mean ± SEM. Results from Fisher’s LSD are displayed. Residuals were observed and three statistical outliers (ROUT Q = 1%) were removed from the mean visit duration in periphery analysis (2 vehicle, 1 PEXI)

Fig. 4

Microglia depletion exacerbates VILI-induced delirium-like behaviors. A The percentage of spontaneous alternations in the Y-maze was significantly lower in PEXI- compared to vehicle-treated VILI mice (n = 8 PEXI, n = 10 vehicle, t = 2.146, p < 0.01), indicating decreased attention and impaired memory. B There is a strong non-statistically significant trend towards less time spend in the open arms of the Elevated Plus Maze in PEXI- compared to vehicle-treated VILI mice (n = 8 PEXI, n = 10 vehicle, t = 1.715, p = 0.09). C Time spent immobile in the Open Field was significantly higher in PEXI- compared to vehicle-treated VILI mice (n = 8 PEXI, n = 10 vehicle, t = 2.364, p = 0.03), also indicating increased anxiety-related behavior. D PEXI-treated VILI mice had a significantly higher coefficient of variation ([standard deviation/mean] × 100, using absolute value of distance in 5-min intervals) compared to vehicle-treated VILI mice (n = 8 PEXI, n = 10 vehicle, t = 2.416, p = 0.02), indicating greater fluctuations in behavior. Two PEXI VILI animals died during ventilation have no post VILI behavior. Animals were excluded from analyses if they did not participate in a specific behavioral assay, exclusion criteria are defined in the Methods section (Open Field: post VILI—2 vehicle, 2 PEXI; Y-maze: post VILI—2 vehicle, 2 PEXI; Elevated Plus Maze: post VILI—2 vehicle, 2 PEXI). Quantitative data are expressed in mean ± SEM. Results from Fisher’s LSD are displayed. Residuals were observed and there were no influential outliers

Fig. 5

A The simple linear regression including vehicle and PEXI-treated mice showed a significant inverse correlation between frontal neuronal CC3%Area and %Spontaneous Alternations on the Y-maze behavioral test (n = 9 PEXI, n = 8 vehicle, R² = 0.51, p < 0.01). A Spearman correlation of the same parameters also showed a significant inverse correlation. (R = − 0.57, p = 0.02). Only mice with both CC3 and Y-Maze data were included. B The simple linear regression including vehicle- and PEXI-treated mice showed a significant positive correlation between hippocampus CC3%Area and Time Immobile in the Open Field test (n = 9 PEXI, n = 9 vehicle, R² = 0.41, p < 0.01). A Spearman correlation of the same parameters also showed a significant positive correlation (R = 0.56, p = 0.02). Only mice with both CC3 and Open Field data were included

Fig. 6

A Timeline of microglia repopulation study. 9 mice were treated with 600 mg/kg PEXI for two weeks before being switched to a vehicle-matched diet for an additional two weeks, and 9 were exclusively fed the vehicle-matched diet for four weeks. B There were no significant differences observed in time spent in the periphery of the Open Field either pre or post VILI between vehicle-treated and microglia repopulated mice (n = 8 repopulation, n = 9 vehicle, t = 0.3081, p = 0.76). C There were no significant differences in the mean visit duration to the periphery zone either pre or post VILI between vehicle-treated and microglia repopulated mice (n = 8 repopulation, n = 9 vehicle, t = 1.546, p = 0.13). D There were no significant differences observed in the percentage of spontaneous alternations in the Y-maze either pre or post VILI between vehicle-treated and microglia repopulated mice. (n = 8 repopulation, n = 8 vehicle, t = 0.2302, p = 0.82). E There were no significant differences observed in time spent in the open arms of the elevated plus maze either pre or post VILI between vehicle-treated and microglia repopulated mice (n = 5 repopulation, n = 6 vehicle, t = 0.6034, p = 0.55). Due to a technical failure in the environment for behavioral testing, all mice tested on one day were excluded from Elevated Plus Maze analysis (pre VILI: 4 vehicle, 3 repopulation; post VILI: 3 vehicle, 3 repopulation). F There were no significant differences observed in time immobile during the Open Field test between vehicle-treated and microglia repopulated VILI mice (n = 8 repopulation, n = 9 vehicle, t = 0.3994, p = 0.69). G There were no significant differences observed in coefficient of variation in the Open Field test between vehicle-treated and microglia repopulated VILI mice (n = 8 repopulation, n = 9 vehicle, t = 1.718, p = 0.1). One VILI animal in the repopulation group died during ventilation and has no post VILI behavior. One VILI animal in the vehicle group did not participate in the Y-Maze test. Quantitative data are expressed in mean ± SEM. Results from Sidak’s post hoc comparisons are displayed