IL-6 Inhibition Reduces Neuronal Injury in a Murine Model of Ventilator-induced Lung Injury

Abstract

Mechanical ventilation is a known risk factor for delirium, a cognitive impairment characterized by dysfunction of the frontal cortex and hippocampus. Although IL-6 is upregulated in mechanical ventilation-induced lung injury (VILI) and may contribute to delirium, it is not known whether the inhibition of systemic IL-6 mitigates delirium-relevant neuropathology. To histologically define neuropathological effects of IL-6 inhibition in an experimental VILI model, VILI was simulated in anesthetized adult mice using a 35 cc/kg tidal volume mechanical ventilation model. There were two control groups, as follow: 1) spontaneously breathing or 2) anesthetized and mechanically ventilated with 10 cc/kg tidal volume to distinguish effects of anesthesia from VILI. Two hours before inducing VILI, mice were treated with either anti-IL-6 antibody, anti-IL-6 receptor antibody, or saline. Neuronal injury, stress, and inflammation were assessed using immunohistochemistry. CC3 (cleaved caspase-3), a neuronal apoptosis marker, was significantly increased in the frontal (P < 0.001) and hippocampal (P < 0.0001) brain regions and accompanied by significant increases in c-Fos and heat shock protein-90 in the frontal cortices of VILI mice compared with control mice (P < 0.001). These findings were not related to cerebral hypoxia, and there was no evidence of irreversible neuronal death. Frontal and hippocampal neuronal CC3 were significantly reduced with anti-IL-6 antibody (P < 0.01 and P < 0.0001, respectively) and anti-IL-6 receptor antibody (P < 0.05 and P < 0.0001, respectively) compared with saline VILI mice. In summary, VILI induces potentially reversible neuronal injury and inflammation in the frontal cortex and hippocampus, which is mitigated with systemic IL-6 inhibition. These data suggest a potentially novel neuroprotective role of systemic IL-6 inhibition that justifies further investigation.

Keywords: COVID-19; IL-6; VILI; delirium; neuronal injury.

Figure 1.

Frontal and hippocampal CC3 (cleaved caspase-3) is significantly elevated in ventilation-induced lung injury (VILI) mice. (A) Schematic of experimental design and representative ROIs of measured brain regions. (B) Quantification of lung inflammation (percentage of PMNs in BALF) (n = 6 in the spontaneous breathing [SB] group, n = 6 in the 10 cc/kg group, and n = 8 in the VILI group). (C and D) Quantification of CC3 in the frontal cortex and the hippocampus, in which each dot represents one animal (n = 6 in the SB group, n = 6 in the 10 cc/kg group, and n = 8 in the VILI group). (E) Representative sections stained for CC3 (positive signal displayed in green) overlaid on DAPI nuclear stain (blue). Magnified regions of an area of frontal neocortex and hippocampus from the micrograph directly above. **P < 0.01 and ****P < 0.0001. BALF = BAL fluid; ns = not significant; PMN = polymorphonuclear cell; ROI = region of interest.

Figure 2.

VILI increases frontal cortical activity, neuronal stress response, and cortical inflammation. (A and B) Quantification of the levels of neuronal activity (c-fos) and of cellular stress response (HSP90 [heat shock protein-90]) within the frontal cortex (A) (n = 6 in the SB group, n = 6 in the 10 cc/kg group, and n = 8 in the VILI group) and hippocampus (B) (n = 6 in the SB group, n = 6 in 10 cc/kg group, and n = 8 in the VILI group). Each dot represents one animal. (C) Representative sections stained for either c-fos or HSP90 (positive signal displayed in green) overlaid on DAPI nuclear stain (blue) for the SB and VILI groups. Magnified regions of a section of the frontal neocortex are inset. (D) IL-6 is significantly increased in VILI brains compared with SB or 10 cc/kg control mice (n = 6 in the SB group, n = 6 in the 10 cc/kg group, and n = 8 in the VILI group). (E) IL-1β is significantly increased in VILI brains compared with SB brains, but there is no significant difference in IL-1β between the 10 cc/kg group and SB group or the 10 cc/kg group and VILI group. (F) TNF-α is significantly increased in VILI group compared with both the 10 cc/kg and SB groups, but there is no significant difference in TNF-α between the SB and 10 cc/kg groups. (G) Representative micrographs stained for IL-6 (green). Of note is the IL-6 staining within the cortical BV in the VILI group that is absent in the SB control group. Cellular morphology (tissue autofluorescence) is displayed in gray, and cell nuclei are revealed by DAPI staining (blue). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. BV = blood vessel.

Figure 3.

Cortical IL-6, IL-1β, and TNF-α positively correlate with neuron injury marker. (A) Pearson’s correlation analyses demonstrate significant positive relationships between cortical CC3 and IL-6, IL-1β, and TNF-α. Animals in the SB group are indicated in blue, and animals in the VILI group are indicated in red. Significance (P) and fitness (r²) values are indicated. (B) Micrographs of frontal cortical neurons stained for IL-6 displayed in green and CC3 displayed in red. Magnified areas of individual neurons and glial cells are inset. Note that CC3-positive neurons also costain for IL-6 in neocortex of VILI animals. *P < 0.05 and **P < 0.01.

Figure 4.

IL-6 inhibition significantly reduces frontal and hippocampal CC3 expression. (A) Schematic of experimental design and timeline. (B) There are no significant differences in oxygen saturations between the three VILI intervention groups (VILI saline, VILI anti–IL-6 antibody, and VILI anti–IL-6 receptor antibody) (n = 6/group). (C) PMNs in BALF are not significantly different between the three VILI groups (n = 6/group). (D) Frontal and hippocampal CC3 expressions are significantly increased in the VILI saline group compared with SB control, but significantly reduced in both VILI IL-6–inhibited groups (n = 5–6/group). One VILI saline animal was excluded because of poor perfusion, resulting in inaccurate analysis. (E) IL-6 is significantly increased in the VILI saline group compared with the SB control group but is significantly reduced in both VILI IL-6–inhibited groups (n = 5–6/group). (F) There is no significant difference in the percentage area of IL-1β between all groups (n = 5–6/group). (G) TNF-α is significantly increased in the VILI saline group compared with the SB control group and is significantly reduced in both VILI IL-6–inhibited groups (n = 5–6/group). (H–J) Pearson’s correlation analysis shows a direct and significant relationship between IL-6 and CC3 and TNF-α and CC3 but no significant relationship between IL-1β and CC3. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.