The cholinergic drug galantamine ameliorates acute and subacute peripheral and brain manifestations of acute respiratory distress syndrome in mice

Abstract

Acute respiratory distress syndrome (ARDS) is a life-threatening form of acute lung injury (ALI), which is a common cause of respiratory failure and high mortality in critically ill patients. Long-term mortality and brain dysfunction have been documented in ARDS patients after hospital discharge. Inflammation plays a key role in ALI/ARDS pathogenesis. Neural cholinergic signaling regulates cytokine responses and inflammation. Here, we studied the effects of galantamine, an approved cholinergic drug (for Alzheimer's disease) on ALI/ARDS severity and inflammation in mice, using a clinically relevant mouse model induced by intratracheal administration of hydrochloric acid and lipopolysaccharide. Mice were treated 30 mins prior to each insult with vehicle or galantamine (4 mg/kg, i.p.). Galantamine treatment significantly decreased bronchoalveolar lavage (BAL) and serum TNF, IL-1β, and IL-6 levels, as well as BAL total protein and myeloperoxidase and lung histopathology in ALI/ARDS mice. In addition, galantamine improved the functional state of mice with ALI/ARDS during a 10-day monitoring and attenuated lung injury and indices of brain inflammation at 10 days. These findings support further studies utilizing this approved cholinergic drug in therapeutic strategies for ARDS and its subacute sequelae.

Keywords: acute lung injury; acute respiratory distress syndrome; brain; cholinergic signaling; galantamine; inflammation.

Figure 1.. Galantamine treatment suppresses pro-inflammatory cytokine levels in mice with ALI/ARDS.

(A) Schematic depiction of the experimental design. Anesthetized mice were administered with HCL and LPS (i.t.) 24h apart and treated 30 mins prior to each insult with galantamine (4 mg/kg) or vehicle administered i.p. Another (control) group of anesthetized mice was subjected to the same experimental procedure but administered vehicle (saline). Mice were euthanized at 30h and blood, bronchoalveolar lavage (BAL), lung and brain (at 10 days) were collected and processed for analysis. (B) BAL TNF levels are significantly higher in mice with ALI/ARDS treated with vehicle (V) (***P=0.0002; Kruskal Wallis test, Dunn’s multiple comparisons test) and in mice with ALI/ARDS treated with galantamine (G) (*P=0.0486) compared with control mice. BAL IL-6 levels are significantly higher in mice with ALI/ARDS treated with vehicle compared with control mice (***P=0.0002; Kruskal Wallis test, Dunn’s multiple comparisons test) and galantamine significantly decreases these levels (*P=0.0226). BAL IL-1β levels are significantly higher in mice with ALI/ARDS treated with vehicle (****P<0.0001; one-way ANOVA, Tukey’s multiple comparisons test) and in mice with ALI/ARDS treated with galantamine (****P<0.0001) compared with control mice. Galantamine significantly decreases IL-1β levels in mice with ALI/ARDS compared with mice treated with vehicle (*P=0.0422). (C) Serum TNF levels are significantly higher in mice with ALI/ARDS treated with vehicle than control mice (****P<0.0001; one-way ANOVA, Tukey’s multiple comparisons test) and galantamine significantly decreases these values (***P=0.0003). Serum IL-6 levels are significantly higher in mice with ALI/ARDS treated with vehicle than control mice (**P=0.0031; one-way ANOVA, Tukey’s multiple comparisons test) and galantamine significantly decreased these values (**P=0.0065). Serum IL-1β levels are significantly higher in mice with ALI/ARDS treated with vehicle than control mice (**P=0.017; Kruskal Wallis test, Dunn’s multiple comparisons test) and galantamine significantly decreases these values (*P=0.0430). Data are represented as individual mouse data points with mean ± SEM. See Materials and Methods for details.

Figure 2.. Galantamine treatment ameliorates markers of acute lung damage in mice with ALI/ARDS.

(A) At 30h BAL total protein levels are significantly higher in mice with ALI/ARDS treated with vehicle (Veh) (****P<0.0001; one-way ANOVA, Tukey’s multiple comparisons test) compared with control mice and galantamine treatment significantly decreases BAL total protein in ALI/ARDS mice (**P=0.0007) to levels no different than controls. (B) BAL MPO levels are significantly higher in mice with ALI/ARDS treated with vehicle than control mice (***P<0.0005; one-way ANOVA, Tukey’s multiple comparisons test) and galantamine significantly decreases these values (***P=0.00125) to levels no different than controls. ((C) Representative H&E images of mouse lung tissue sections show the degree of ALI injury in the two-hit model, including neutrophil infiltration in alveolar space (yellow arrowheads) and interstitial space (red arrowheads). Top row images, scale bar = 20 μm; bottom row images, scale bar = 10 μm. (D) The acute lung injury score in mice with ALI/ARDS treated with vehicle (****P<0.0001; one-way ANOVA, Tukey’s multiple comparisons test) and those treated with galantamine (****P<0.0001) is significantly higher compared with controls. Galantamine treatment of mice with ALI/ARDS significantly decreases the score compared with vehicle treatment (****P<0.0001). Each individual point represents an average of 5 sections that were scored. Data are represented as individual mouse data points with mean ± SEM. See Materials and Methods for details.

Figure 3.. Galantamine treatment improves the functional activity of mice with ALI/ARDS with no effect on body weight.

(A) Mice with ALI/ARDS treated with vehicle (n=14) or galantamine (n=14) have impaired functional activity indicated by high illness scores compared with controls (n=8) (***P<0.0001; repeated measures two-way ANOVA, Tukey’s multiple comparisons test) during 10-day monitoring. Galantamine treatment improves the functional activity of mice with AIL/ARDS reflecting a significantly lower illness score compared with vehicle treatment (***P<0.0001). (B) The overall body weight of mice with ALI/ARDS treated with vehicle (n=14) is significantly lower compared with controls (n=8) (**P=0.0058; repeated measures two-way ANOVA, Tukey’s multiple comparisons test) and not significantly different than ALI/ARDS mice treated with galantamine during 10-day monitoring. See Materials and Methods for details.

Figure 4.. Galantamine treatment attenuates indices of subacute ALI/ARDS in mice.

(A) BAL total protein levels are significantly higher in mice with ALI/ARDS treated with vehicle (Veh) (****P=0.0014; one-way ANOVA, Tukey’s multiple comparisons test) compared with control mice and not significantly different than ALI/ARDS mice treated with galantamine. (B) BAL MPO levels are significantly higher in mice with ALI/ARDS treated with vehicle than control mice (***P=0.0199; one-way ANOVA, Tukey’s multiple comparisons test) and not significantly different than ALI/ARDS mice treated with galantamine. (C) Representative H&E images of mouse lung tissue sections show the degree of ALI injury in the two-hit model after 10 days, including neutrophil infiltration in interstitial space (red arrowheads) and thickening of hyaline membranes (green arrowheads). Top row images, scale bar = 20 μm; bottom row images, scale bar = 10 μm. (D) The lung injury score in mice with ALI/ARDS treated with vehicle is significantly higher compared with controls (****P<0.0001; Kruskal Wallis test, Dunn’s multiple comparisons test). Galantamine treatment of mice with ALI/ARDS significantly decreases the score compared with vehicle treatment (**P<0.0099). Each individual point represents an average of 5 sections that were scored. Data are represented as individual mouse data points with mean ± SEM. See Materials and Methods for details.

Figure 5.. Galantamine treatment decreases microglial accumulation in the hippocampus of mice with subacute ALI/ARDS.

(A) Representative images of IBA1 staining of brain (hippocampus) shown in wide tiles (Scale bar = 200 μm) and with 20x magnification (Scale bar = 50 μm in the three groups of mice. (B) The number of microglia in the hippocampus of mice with ALI/ARDS treated with vehicle is significantly higher compared with controls (***P=0.0008; one-way ANOVA, Tukey’s multiple comparisons test). Galantamine treatment of mice with ALI/ARDS significantly decreases microglial accumulation in the hippocampus compared with vehicle treatment (*P=0.0181). (C) The ramification index of microglia in the hippocampus of mice with ALI/ARDS treated with vehicle is significantly higher compared with controls (**P=0.001; one-way ANOVA, Tukey’s multiple comparisons test). Galantamine treatment of mice with ALI/ARDS significantly decreases the microglial ramification index in the hippocampus compared with vehicle treatment (*P=0.029). Data are represented as individual mouse data points with mean ± SEM. See Materials and Methods for details.