Intranasal versus intratracheal exposure to lipopolysaccharides in a murine model of acute respiratory distress syndrome

Abstract

Due to frequent and often severe lung affections caused by COVID-19, murine models of acute respiratory distress syndrome (ARDS) are increasingly used in experimental lung research. The one induced by a single lipopolysaccharide (LPS) exposure is practical. However, whether it is preferable to administer LPS intranasally or intratracheally remains an open question. Herein, female C57Bl/6 J mice were exposed intranasally or intratracheally to one dose of either saline or 3 mg/kg of LPS. They were studied 24 h later. The groups treated with LPS, either intranasally or intratracheally, exhibited a pronounced neutrophilic inflammation, signs of lung tissue damage and protein extravasation into the alveoli, and mild lung dysfunction. The magnitude of the response was generally not different between groups exposed intranasally versus intratracheally. However, the variability of some the responses was smaller in the LPS-treated groups exposed intranasally versus intratracheally. Notably, the saline-treated mice exposed intratracheally demonstrated a mild neutrophilic inflammation and alterations of the airway epithelium. We conclude that an intranasal exposure is as effective as an intratracheal exposure in a murine model of ARDS induced by LPS. Additionally, the groups exposed intranasally demonstrated less variability in the responses to LPS and less complications associated with the sham procedure.

Figure 1

Schematic of the experimental protocol. On day 1, the mice were assigned to one of four experimental groups: 1- saline delivered intranasally; 2- saline delivered intratracheally; 3- LPS delivered intranasally; or 4- LPS delivered intratracheally. On day 2 (in gray), exactly 24 h after saline or LPS exposure, the mice were tested. Respiratory mechanics was assessed with the flexiVent on 2 mice in each group. The bronchoalveolar lavages, the lung wet-to-dry weight ratio, and the histology on lung slices were performed on all mice. The contractile assays with excised tracheas were performed on one mouse tested with the flexiVent and one mouse not tested with the flexiVent in each group. This protocol was repeated 6 times to reach the sample sizes shown in Table 1.

Figure 2

Cellular lung inflammation in the bronchoalveolar lavages. (A) Total cells per mL of BAL, and the percentage of (B) macrophages and (C) neutrophils in BAL are depicted for mice treated with either saline (black) or LPS (grey) through either the intranasal (IN) or the intratracheal (IT) route. The results of the two-way ANOVA are shown at the bottom of each graph. *Indicates statistically significant from the IN-exposed group treated with saline. †Indicates statistically significant from the group treated with saline exposed through the same route. n = 18 per group and the data shown are means ± SD.

Figure 3

Cellular inflammatory infiltrates into the parenchymal tissue. The upper panels are representative lung sections derived from mice treated with either saline (top) or LPS (bottom) through either the intranasal (IN) or the intratracheal (IT) route. The bar graph below depicts the mean inflammatory score in each group. Scale bars are 100 µm. The results of the two-way ANOVA are shown at the bottom of the graph. *Indicates statistically significant from the IN-exposed group treated with saline. †Indicates statistically significant from the group treated with saline exposed through the same route. n = 18 per group and the data shown are means ± SD.

Figure 4

Number of goblet cells per mm of basement membrane. The upper panels are representative lung sections derived from mice treated with either saline (top) or LPS (bottom) through either the intranasal (IN) or the intratracheal (IT) route. The bar graph below depicts the mean in each group. Scale bars are 25 µm. The results of the two-way ANOVA are shown at the bottom of the graph. *Indicates statistically significant from the IN-exposed group treated with LPS. n = 18 per group and the data shown are means ± SD.

Figure 5

Protein extravasation into the alveoli. The concentration of (A) total proteins and (B) albumin in bronchoalveolar lavage fluid (BALF) are depicted for mice treated with either saline (black) or LPS (grey) through either the intranasal (IN) or the intratracheal (IT) route. The results of the two-way ANOVA are shown at the bottom of each graph. †Indicates statistically significant from the group treated with saline exposed through the same route. n = 18 per group and the data shown are means ± SD.

Figure 6

Respiratory mechanics. (A) The quasi-static elastance of the respiratory system (E_st) and (B) A (a proxy for the inspiratory capacity) are depicted for mice treated with either saline (black) or LPS (grey) through either the intranasal (IN) or the intratracheal (IT) route. The results of the two-way ANOVA are shown at the bottom of each graph. n = 12 per group and the data shown are means ± SD.

Figure 7

Contractility of excised tracheas. The maximal force generated by excised tracheas in response to (A) a cumulative concentration of methacholine (MCh) ending with 10^–4 M or (B) a cumulative concentration of potassium chloride (KCl) ending with 160 mM are depicted for mice treated in vivo with either saline (black) or LPS (grey) through either the intranasal (IN) or the intratracheal (IT) route. The results of the two-way ANOVA are shown at the bottom of each graph. n = 12 per group and the data shown are means ± SD.