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

Journal of Neuroinflammation volume 21, Article number: 270 (2024) Cite this article

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.

Background

Delirium is a highly prevalent clinical condition that affects 50–85% of patients on mechanical ventilation and is associated with increased mortality, prolonged hospitalization, and a three-fold increased risk of dementia [1,2,3,4,5]. Acute frontal and hippocampal neuronal injury are thought to underlie the clinical manifestations of delirium, which include a fluctuating course of inattention, executive dysfunction, or short-term memory impairment [1,2,3].

We previously showed a central pathological role for peripheral interleukin-6 (IL-6) in mediating neuronal and functional delirium-like phenotypes via the IL-6 trans-signaling pathway in murine models of ventilator-induced acute lung injury (VILI) [6, 7] and urinary tract infection [8] while others have demonstrated a pathogenic role for IL-6 in mediating neurocognitive decline in an orthogonal model of perioperative delirium [9]. We recently showed that use of tocilizumab, an IL-6 pathway inhibitor, was associated with less delirium and coma in critically ill patients with COVID-19 [10]. In the IL-6 trans-signaling pathway, IL-6 complexes with soluble IL-6 receptor in the periphery and directly induces neuronal injury without needing to engage any classic membrane receptors, which neurons lack but microglia, the brain’s resident macrophage cells, express. Although microglia regulate neuronal function in response to acute systemic inflammatory injuries, both neurotoxic and neuroprotective microglia phenotypes have been reported [11,12,13,14] and it remains unknown whether microglia activated by mechanical ventilation ameliorate or exacerbate VILI-induced neuronal injury.

Accordingly, in this study, we seek to clarify the role of microglia on structural and functional delirium-like phenotypes in VILI. As prior studies have reported a neuroprotective role for microglia in mitigating systemic inflammation-induced brain injury [15], in this study we hypothesize that in a model of short-term VILI, microglia play a neuroprotective role to ameliorate delirium-like phenotypes.

Methods

Microglia depletion

Microglia were depleted using a colony stimulating factor 1 receptor (CSF1R) inhibitor, Pexidartinib/PLX 3397 (PEXI) (MedChemExpress, Monmouth Junction, NJ, USA), that has previously been demonstrated to effectively deplete resident microglia in the mouse brain [16]. Microglia depletion was accomplished by feeding mice chow formulated with 600 mg/kg PEXI over a two-week period, as demonstrated in prior studies [16], and confirmed using Iba-1 staining.

Microglia repopulation

Prior studies have shown that microglia are restored to untreated levels and morphologies 14 days after discontinuation of PEXI treatment [16]. In our study, microglia repopulation was accomplished by switching mice to the vehicle diet for 2 weeks after 2 weeks of PEXI diet, while the control group received vehicle diet for a total of 4 weeks.

Animals

For the initial experiment, 36 C57BL/6 J male mice aged 4–9 months were assigned to one of two diets, and within the diets they were assigned to either VILI or spontaneous breathing (SB) groups: 600 mg/kg PEXI (n = 5 SB, 13 VILI) and vehicle-matched control (n = 6 SB, 12 VILI) for two weeks each (Fig. 1A). For the repopulation experiments, 28 C57BL/6 J male mice aged 4–9 months were assigned to a vehicle (n = 5 SB, 9 VILI) or a repopulation group (n = 5, SB, 9 VILI). The repopulation group was fed the PEXI diet for 2 weeks and switched to the vehicle-matched diet for 2 weeks, while the vehicle group was fed the vehicle-matched diet for the total 4-week period (Fig. 6A). Mouse weights were measured twice weekly while on the diet to assess change in body mass. All mice were housed in Cedars-Sinai’s AAALAC accredited animal facility under standard conditions (kept in ventilated cages at approximately 21 °C, 40–70% humidity, a 12-h light/dark cycle, with food and water available to the animals ad libitum). All procedures herein follow the recommendations in the ARRIVE guidelines for research involving the use of animals. All experiments were conducted in accordance with Cedars-Sinai Medical Center Institutional Animal Care and Use Committee (IACUC) guidelines under an approved protocol (#7914).

Fig. 1
figure 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)

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VILI model

VILI was induced as described in our prior work [6, 17]. Briefly, mice were anesthetized with a mixture of 75 mg/kg ketamine and 0.5 mg/kg dexmedetomidine injected intraperitoneally before being orotracheally intubated. VILI was induced via ambient room air volume-controlled mechanical ventilation using a high tidal volume (35 cc/kg) for 2 h in the supine position, using VentElite Small Animal Ventilators (Harvard Apparatus, Holliston, MA, USA). Respiratory rate was set at 70 breaths/min with zero positive end-expiratory pressure. Body temperature was maintained using a 38 ℃ heating pad (Hallowell EMC, Pittsfield, MA, USA), and oxygen saturations were monitored periodically using MouseOx Plus (STARR Life Sciences, Oakmont, PA, USA). Immediately before intubation, 0.5 mL saline was administered subcutaneously to maintain hydration and the eyes of mice were protected with a thin coat of Paralube (Dechra Pharmaceuticals, Northwich UK). Anesthesia was reversed with 1.5 mg/kg atipamezole.

Behavioral testing

The Open Field, Elevated Plus Maze, and Y-maze were used to evaluate delirium-like behaviors in all mice 1 day before and 15 h after induction of VILI, as previously described [18]. Brief descriptions of these assays follow below:

Open Field: The Open Field test largely measures exploratory behavior and locomotor activity, which can be affected by delirium-like phenotypes including altered level of consciousness, anxiety, and cognitive decline [19, 20]. Individual mice were placed in opaque arenas (40 × 40 × 40 cm) and recorded for 45 min with a computer-operated camera (Stoelting Digital USB 2.0 CMOS Camera). The apparatus was divided into a 20 × 20 cm center zone and surrounding periphery zone for analysis. Measurements of total distance travelled (meters), distance travelled in each zone (meters) and time spent in each zone (seconds) were collected and analyzed using ANY-maze Video Tracking Software (Stoelting Co., Wood Dale, IL, USA). In uninjured mice, locomotor activity decreases over time and is less variable due to habituation to the testing environment. Failure to habituate to the testing environment indicates the presence of altered consciousness. For each mouse the distance moved (meters) in the open field across the 45-min test was quantified and binned in 5-min intervals, which were used to calculate the coefficient of variation ([standard deviation/mean] × 100), as a marker of fluctuating behavior. Time spent immobile, which has been shown to be increased in anxiety-related conditions [21], were generated by the ANY-maze software.

Elevated Plus Maze: The Elevated Plus Maze assesses anxiety-related behavior and altered level of consciousness [20, 22]. The apparatus is elevated 30 cm above the floor, consisting of two opposing 310 mm long closed arms surrounded by walls and two opposing 310 mm long open arms without walls creating the appearance of a plus sign (+) from above. Mice exhibiting anxiety behavior will avoid the open arms in favor of the walled closed arms. Individual mice were placed near the center of apparatus facing an open arm and allowed to move freely while being recorded for 5 min. Time spent in open and closed arms was collected and analyzed.

Y-maze: The Y-maze tests multiple types of memory including short-term, attentional, and cognitive [23, 24]. The apparatus consists of three walled arms of 360 mm length each at an angle of 120 degrees from each other. Mice are placed into one arm and are recorded for 10 min. Uninjured mice typically prefer to explore a new arm of the Y-maze rather than one that was recently visited, so repeated exploration of the same arms indicates impaired memory. ANY-maze tracking software was used to measure number and sequence of arm entries, and the percentage of spontaneous alternations was calculated.

Histology

Brain isolation

After administering ketamine/dexmedetomidine anesthetic, mice were perfused with 20 mL PBS containing 0.5 mM ethylenediaminetetraacetic acid. The right hemisphere was collected and fixed in PBS buffered 4% paraformaldehyde on ice for 30 min before sucrose was added to cryoprotect the tissue in a 2% paraformaldehyde + 30% sucrose solution. After 1 week, 30-μm coronal cryosections were prepared and stored free-floating at 4 °C in PBS + 0.02% sodium azide. Plasma IL-6 concentrations were quantified by ELISA (Cat #: M6000B, R&D Systems, Minneapolis, MN).

Immunohistochemistry and microscopy

Sections underwent heat-induced epitope retrieval for 10 min in 6.0 pH citrate antigen retrieval buffer before permeabilizing and blocking in a blocking solution at room temperature. Sections were then immersed in primary antibody solution overnight containing antibodies for cleaved caspase-3 (CC3), IL-6, Iba1, NeuN, and antibody diluent (see Supplementary Table 1 for specific configurations). After primary antibody solution was removed, fluorescent secondary antibodies were added to the tissue. CC3 signal was amplified using Opal 650 reagent (Akoya Biosciences, Marlborough, MA, USA). Sections were then imaged using Carl Zeiss AxioImager Z.2 epi-fluorescence microscope 10 × objective lens as a tiled image that included the frontal cortex and hippocampus.

Image analysis

Images were exported as 8-bit TIFF files and analyzed using ImageJ. Three coronal sections per animal were analyzed by drawing regions of interest around 1.2 mm2 of the frontal cortex or the entirety of the hippocampus and setting a threshold using the pixel intensity histogram. The number of positive pixels at that threshold was measured as a percent area of the total regions of interest, and results from the three sections were averaged for each animal.

Statistical analysis

Prism (GraphPad Software, Boston, MA, USA) was used for statistical analyses. Differences between groups in histological analyses (Fig. 2) were evaluated using independent sample Welch’s t-tests. Animals were excluded from histological analysis in cases of death or when less than two sections were quantifiable due to technical failures. Behavioral tests (Figs. 3, 4, 6) were analyzed using two-way repeated measures ANOVAs, or a mixed effects model whenever values were missing from exclusion or death. To account for VILI’s significant injury effects, animals were not included in behavioral analyses in cases of death, technical failures, or nonparticipation in a behavioral test. Nonparticipation was defined as moving ≤ 5 m over 45 min in the open field test, failing to move after first arm entry in Elevated Plus Maze, or failing to enter at least two arms of the Y-maze throughout the entire test duration. Animals that did not participate in a behavioral assay were excluded from the analysis of all data derived from that assay. Post-hoc comparisons were evaluated with an uncorrected Fisher’s LSD test due to the exploratory nature of these analyses. Analyses of complete datasets without any exclusions are available in the Supplementary Information. Figure legends include specific information on data exclusion for the corresponding analysis.

Fig. 2
figure 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

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Fig. 3
figure 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)

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Fig. 4
figure 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

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Results

Microglia depletion significantly worsens VILI-induced neuronal injury

We first established that frontal/hippocampal neuronal CC3 or IL-6 were not significantly elevated at baseline in spontaneously breathing PEXI- compared to vehicle-treated mice (Supplemental Fig. 1).

The experimental timeline is shown in Fig. 1A. There were no significant differences in oxygen saturations, polymorphonuclear cells (PMNs), or plasma IL-6 between vehicle- and PEXI- treated VILI animals (Fig. 1B–D). Frontal and hippocampal neuronal CC3 levels were significantly higher in PEXI- compared to vehicle-treated VILI mice (Fig. 2A, B; p < 0.01 and p < 0.01, respectively). PEXI-treated animals demonstrated significantly reduced IBA-1 signal compared to the vehicle-treated mice, indicating over 80% depletion of microglia in the cortex and hippocampus (Fig. 2C, D; p < 0.01 and p < 0.01, respectively). Representative fluorescence microscopy images for frontal/hippocampal neuronal CC3 for vehicle- and PEXI-treated VILI are shown in Fig. 2E. Figure 2F shows representative Iba-1 fluorescence microscopy images of vehicle and PEXI-treated mice, respectively. Representative 20 × fluorescence microscopy images for vehicle and PEXI-treated CC3 and IL-6 are shown in Fig. 2G. Following VILI, frontal IL-6 levels were significantly higher in PEXI-compared to vehicle-treated animals (Fig. 2H; p < 0.01).

Microglia depletion significantly worsens delirium-like behaviors after VILI

There were no significant differences in the baseline behavioral assessments prior to VILI induction between PEXI- and vehicle-treated mice (Figs. 3, 4), indicating that microglia depletion did not affect behavior, as has been previously reported [16]. Mice spent significantly more time in the periphery zone of the Open Field maze after induction of VILI, as we have previously reported [7]. PEXI-treated VILI mice demonstrated significantly increased time in the periphery zone compared to vehicle-treated VILI controls (Fig. 3A; p = 0.01). PEXI-treated VILI mice also demonstrated significantly increased mean visit duration to the periphery zone (Fig. 3B, p < 0.01), despite no significant differences in entries to the peripheral zone (Supplemental Fig. 3). Representative heat maps from the Open Field are shown in Fig. 3C, D.

In Y-maze, PEXI-treated VILI mice demonstrated significantly lower percentage of spontaneous alternations compared to vehicle-treated VILI controls (Fig. 4A; p < 0.01). PEXI-treated VILI mice demonstrated a trend towards less time in the open arms of Elevated Plus Maze compared to vehicle-treated VILI controls (Fig. 4B; p = 0.09). PEXI-treated VILI mice also demonstrated significantly increased time spent immobile in the Open Field (Fig. 4C; p = 0.03). The coefficient of variation ([standard deviation/mean] × 100, using absolute value of distance in 5-min intervals) was significantly higher in PEXI-treated VILI mice compared to vehicle-treated VILI controls, suggesting increased fluctuating behavior in the microglia depleted mice (Fig. 4D; p = 0.02). Statistics summarizing factors and interactions are shown in Supplemental Table 1.

Neuronal CC3 correlates with delirium-like behaviors

There was a significant inverse correlation between frontal neuronal CC3 and performance on the Y-maze test after VILI in mice from both groups (Fig. 5A; R2 = 0.51, p < 0.01). There was also a significant correlation between neuronal CC3 in the hippocampus and time spent immobile in the open field test after VILI in mice from both groups (Fig. 5B; R2 = 0.41, p < 0.01). There were non-statistically significant trends towards an inverse correlation between hippocampal neuronal CC3 and spontaneous alternations on Y-maze as well as a positive correlation between hippocampal neuronal CC3 and time spent in the periphery zone of the Open Field apparatus (Supplemental Fig. 2). These data support the role of neuronal CC3 as a candidate neuronal marker of delirium-like behavior, as we have previously reported [18].

Fig. 5
figure 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

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Microglia repopulation restores delirium-like behaviors after VILI

To confirm that microglia depletion worsened delirium-like behavior in VILI mice, we carried out a microglia repopulation experiment. Mice were fed PEXI diet for 2 weeks, then switched to regular chow for another 2 weeks, prior to induction of VILI and behavioral testing. There were no significant differences in delirium-like behaviors between microglia repopulated and vehicle-treated VILI mice (Fig. 6). Specifically, there were no differences between microglia repopulated and vehicle-treated mice in time spent in the periphery of the Open Field, mean visit duration to the periphery zone, percentage of spontaneous alternation in the Y-maze, time spent in the open arms of the Elevated Plus Maze, time spent immobile, or the coefficient of variation (Fig. 6B–G). Statistics summarizing factors and interactions are shown in Supplemental Table 1.

Fig. 6
figure 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

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Discussion

In this study, we demonstrate 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.

VILI is an exceedingly common etiology of delirium that affects millions of patients every year [2, 25,26,27]. The health impact of VILI-induced delirium is expected to increase substantially in the coming years as our population ages and requires greater use of critical care interventions. Furthermore, epidemiological and clinical data support a causal relationship between delirium and dementia and suggest that delirium mitigation may be a rare and much-needed opportunity to ameliorate the prevalence of dementia [4, 28, 29]. Our study’s findings suggest that manipulation of microglia physiology in the acute phase of VILI with emerging microglia-targeted treatments may provide a means to mitigating delirium, and thus perhaps incipient dementia [30].

Prior studies have reported that microglia exist in various proportions of neuroprotective or neurotoxic phenotypes, with the latter prevailing in neurodegenerative conditions such as dementia [13, 16, 31,32,33,34]. In this study, we demonstrate a protective role for microglia in mitigating the neurological effects of VILI in relatively young and healthy mice; however, future studies are needed to evaluate the role of potentially neurotoxic microglia in neurodegenerative substrates that may lower the threshold for delirium. The acute nature of our VILI model may explain why we observe a protective rather than deleterious effect that has been reported in studies on sepsis and post-operative models that evaluated effects of microglia depletion on cognitive function 3 days after injury [35, 36]. In contrast, we utilized a relatively short-term, 2-h model of VILI though patients are frequently subjected to the effects of VILI for much longer durations. Whether prolonged exposure of the brain to the effects of VILI alter microglia physiology to neurotoxic phenotypes remains unknown and is indicated for evaluation in future studies. Furthermore, while our analysis of microglia morphology (Supplemental Fig. 4) indicates that VILI increases the proportion of high activity microglia, additional studies are needed to clarify the exact timeline of these morphological changes and the associated functional changes [37, 38].

This study’s findings are consistent with prior studies that show a deleterious effect of peripheral, lung-derived IL-6, on delirium-like behaviors and cognitive impairment [6, 7, 17, 39], which may be exacerbated or prolonged by the absence of microglia. There are several mechanisms by which microglia could have the observed neuroprotective effect in our VILI model: (1) microglia release brain-derived neurotrophic factor, which is a key component of neuronal growth, plasticity, and survival, and synaptic function [5, 11]; further BDNF has been reported to have anti-inflammatory properties by modulating release of IL-6 [40] (2) microglia restrict the release of inflammatory factors such as IL-6 by astrocytes [41], (3) inflammation-induced microglial activation is protective of the blood–brain barrier in the acute inflammatory response while sustained inflammation damages the blood–brain barrier [42], (4) neuroprotective microglia express arginase 1, which competes with inducible nitric oxide synthetase over arginine, and reduces neuroinflammation caused by nitric oxide levels [43]. These mechanisms need to be explored in future studies to gain a better understanding of how microglia can be beneficial or detrimental in different stages of acute neuroinflammation.

Our study has several key strengths including the use of a novel model of VILI-induced delirium that encompasses both structural and functional phenotypes. The use of several behavioral tests and the directional consistency of their findings strengthens confidence in the assays as reliable measures of acute delirium-like behaviors. However, our study is not without limitations. The use of a 35 cc/kg tidal volume to induce acute lung injury may not reflect current clinical practice where low tidal volume mechanical ventilation is considered standard of care [44]. However, unlike in many mechanically ventilated patients, our animals did not have any concurrent lung injury conditions such as pneumonia, which could exacerbate VILI effects. Furthermore, we have previously demonstrated beneficial neurological effects of systemic immunomodulation with 35 cc/kg tidal volume, lending target validity to the model [6, 7, 45]. Our study additionally used only male mice to reflect male sex as a risk factor for delirium in the context of mechanical ventilation [46, 47]. Prior studies have also shown more efficient and consistent microglia depletion from PEXI in male compared to female mice [32, 48,49,50]. However, future studies should evaluate whether sex differences affect microglial function in delirium. Moreover, while we demonstrate correlations between CC3 and delirium-like behaviors, we do not provide a full analysis of the significance of CC3’s role in delirium pathophysiology, including activation of any potential apoptotic cascades [51, 52]. Indeed, we have previously shown that CC3 activation can occur without irreversible cellular death [6,7,8] while others have implicated CC3 in both and apoptotic and regulatory pathways [53]. Future studies are needed to clarify to what extent these pathways are implicated in delirium pathogenesis. Finally, we recognize the challenges of evaluating delirium-like behavior in mice following VILI, which represents a significant injury that can lead to reduced locomotor activity. The challenge is compounded by the broad phenomenological manifestation of delirium, which includes hypoactivity [54,55,56,57]. Our behavioral results are nevertheless directionally consistent across different assays and supported by structural CC3 changes in the brain. Furthermore, we believe our model is translationally relevant as our previously published findings have been validated in clinical studies [7, 39].

In summary, in this study we demonstrate a neuroprotective role for microglia in ameliorating neuronal and functional delirium-like phenotypes after VILI. These findings call for consideration of interventions that leverage endogenous microglia physiology to ameliorate delirium and its associated effects.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

CC3:

Cleaved caspse-3

VILI:

Ventilator-induced lung injury

CSF1R:

Colony stimulating factor 1 receptor

PEXI:

Pexidartinib

IL-6:

Interleukin-6

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Acknowledgements

Experimental timelines and behavioral apparatus diagrams created with BioRender.com.

Funding

F. Widjaja Foundation.

Author information

Author notes

  1. Landon Scott and Kevin D. Winzey contributed equally to this work.

Authors and Affiliations

  1. Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Landon Scott, Kevin D. Winzey, Debbie Moreira, Warren G. Tourtellotte & Shouri Lahiri

  2. Biostatistics Shared Resources, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Catherine Bresee

  3. Department of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Jean‑Philippe Vit, Warren G. Tourtellotte & Shouri Lahiri

  4. Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Warren G. Tourtellotte

  5. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    S. Ananth Karumanchi

  6. Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Warren G. Tourtellotte & Shouri Lahiri

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Contributions

LS, KDW, DM, and WGT contributed to the acquisition of data. LS, KDW, CB, JV, SAK, and SL did the analysis and interpretation of the data. LS, KDW, SAK, SL drafted the manuscript. SL and SAK contributed to the study concept and design. SL did the study supervision. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shouri Lahiri.

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Ethics approval and consent to participate

All experiments were conducted in accordance with Cedars-Sinai Medical Center Institutional Animal Care and Use Committee (IACUC) guidelines under an approved protocol and complied with current US law.

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The authors declare no competing interests.

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Scott, L., Winzey, K.D., Moreira, D. et al. Microglia ameliorate delirium-like phenotypes in a murine model of acute ventilator-induced lung injury. J Neuroinflammation 21, 270 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12974-024-03260-y

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Keywords

Journal of Neuroinflammation

ISSN: 1742-2094