Pulmonary permeability assessed by fluorescent-labeled dextran instilled intranasally into mice with LPS-induced acute lung injury

Abstract

Background: Several different methods have been used to assess pulmonary permeability in response to acute lung injury (ALI). However, these methods often involve complicated procedures and algorithms that are difficult to precisely control. The purpose of the current study is to establish a feasible method to evaluate alterations in lung permeability by instilling fluorescently labeled dextran (FITC-Dextran) intranasally.

Methods/principal findings: For the mouse model of direct ALI, lipopolysaccharide (LPS) was administered intranasally. FITC-Dextran was instilled intranasally one hour before the mice were euthanized. Plasma fluorescence intensities from the LPS group were significantly higher than in the control group. To determine the reliability and reproducibility of the procedure, we also measured the lung wet-to-dry weight ratio, the protein concentration of the bronchoalveolar lavage fluid, tight and adherens junction markers and pathological changes. Consistent results were observed when the LPS group was compared with the control group. Simultaneously, we found that the concentration of plasma FITC-Dextran was LPS dose-dependent. The concentration of plasma FITC-Dextran also increased with initial intranasal FITC-Dextran doses. Furthermore, increased fluorescence intensity of plasma FITC-Dextran was found in the intraperitoneally LPS-induced ALI model.

Conclusion/significance: In conclusion, the measurement of FITC-Dextran in plasma after intranasal instillation is a simple, reliable, and reproducible method to evaluate lung permeability alterations in vivo. The concentration of FITC-Dextran in the plasma may be useful as a potential peripheral biomarker of ALI in experimental clinical studies.

Figure 1. H&E staining shows pathological alterations that are characteristic of acute lung injury at 6 h after LPS instillation.

A: The gross appearance showed yellow FITC-Dextran on the pulmonary surface after i.n. instillation with 10 mg of FITC-Dextran/kg body weight (b.w.) for 1 hour. The lungs of the LPS model group showed red dots and swelling. B: Representative normal lung histology. C: Lung edema (arrow) and alveolar wall thickening (arrow head) in the ALI mice. D: Infiltration of many inflammatory cells (arrow shows neutrophils) in the ALI mice induced with i.n. instillation with 0.5 mg of LPS/kg b.w. for 6 h (n = 4). B, C, D the magnification is 400X.

Figure 2. Pulmonary permeability assessed by FITC-Dextran via i.n. instillation.

A: Lung permeability assay using 10 mg of FITC-Dextran kg b.w. via i.n. instillation in the ALI induced by different doses (0.5, 2 and 4 mg/kg b.w.) of LPS by i.n. instillation (n = 3). The fluorescence intensity (FI) of the FITC-Dextran in the plasma showed a dose-dependent increase following the different doses of LPS. B: Lung permeability assay using different doses (1, 3, 15 and 30 mg/kg b.w.) of FITC-Dextran via i.n. instillation in the ALI induced by 0.5 mg of LPS/kg b.w. by intranasal instillation (n = 3, *P<0.05, **P<0.01).

Figure 3. Lung permeability evaluation in the i.p. or i.n. LPS-induced ALI model by FITC-Albumin and FITC-Dextran assay.

A: The FI value of plasma FITC-Albumin (5 mg/kg b.w., via i.n. instillation) in the ALI induced i.n. by LPS (0.5 mg/kg b.w.). B: Similar fold changes were found between FITC-Dextran (10 mg/kg b.w., via i.n. instillation) and EB (20 mg/kg b.w., via i.v. instillation) in the 6 h and 24 h LPS (4 mg/kg b.w., via i.n. instillation) groups compared to the control groups. C: Lung inflammatory lesions and thickened alveolar septa observed in ALI induced by LPS i.p. (15 mg/kg b.w.) (200X magnification). D: The concentration of plasma FITC-Dextran is significantly increased in the LPS i.p. group compared to the control group, with 10 mg of FITC-Dextran/kg b.w. via i.n. instillation (n = 3, *P<0.05, **P<0.01).

Figure 4. Lung permeability evaluation by traditional methods.

A: Protein concentration in the BALF. B: The lung wet-to-dry weight ratios in mice with i.n. instillation with 0.5 mg of LPS/kg b.w. C: PMN count in the lung interstitium. D: PMN count in the BALF of mice with i.n. instillation with 0.5 mg of LPS/kg b.w. (n = 4, *P<0.05; **P<0.01).