Intratracheal Bleomycin-Induced Pulmonary Fibrosis

Description

The intratracheal (IT) bleomycin-induced pulmonary fibrosis model is the standard model of human lung fibrosis. After being anesthetized, animals have a cannula inserted into the trachea and down into the lungs and then bleomycin is slowly infused into the lungs. Animals are evaluated daily for body weight and respiratory status. After animals are sacrificed, lung weight, collagen content and fibrosis scores are determined.

Model Systems
  • C57Bl/6 Mice
Standard Readouts
  • Percent weight change
  • Lung weight
  • Terminal histopathology and/or IHC analyses
  • Lung mechanics
  • Lung Collagen Content
Additional Readouts
  • BAL total and differential cell counts
  • BAL fluid and lung cytokines
  • Lung immunophenotyping
  • Custom assays
Representative DataClick Image to Enlarge
IT Bleomycin-Induced Pulmonary Fibrosis Induction Protocol

C57Bl/6 mice are administered Bleomycin once on Day 0 by intratracheal route. Endpoints are assessed on Day 21.

Lung Pathology Scoring in IT Bleomycin-Induced Pulmonary Fibrosis

Pathology assessment of Day 21 lungs from IT-Bleomycin administered animals. Ashcroft score was determined in H&E-stained tissue using a modified Ashcroft Scale graded from 0-8. Fibrosis score was determined in Masson’s Trichrome-stained tissue using a 0-5 semi-quantitative scoring scale of area staining positive for collagen. Infiltrate score was determined in H&E-stained tissue using a 0-5 semi-quantitative scoring scale based on severity of mononuclear cell infiltration. Images are from animals receiving saline or 3.0 U/kg Bleomycin. Histopathology, immunohistochemistry, and analysis were performed by Dallas Tissue Research. (*p<0.05 compared to the saline-control)

Fibrosis Quantification in IT Bleomycin-Induced Pulmonary Fibrosis

Alpha smooth muscle actin (αSMA) and Collagen I + III Immunohistochemistry in Day 21 lungs from IT-Bleomycin administered animals. Whole slide images were used for global and regional αSMA quantification using a deep learning algorithm. Images are from animals administered either saline or 3.0 U/kg Bleomycin. Histopathology, immunohistochemistry, and analysis were performed by Dallas Tissue Research.

Wet Lung Weight in IT Bleomycin-Induced Pulmonary Fibrosis

Wet lung weight and wet lung weight normalized to Day 21 body weight of Day 21 lungs from IT-Bleomycin administered animals.

Body Weight Change in IT Bleomycin-Induced Pulmonary Fibrosis

IT-Bleomycin administered animals are weighed daily, and body weight compared to Day 0 is calculated. The AUC is calculated to compare treatment arms and is shown in the inset. (*p<0.05 compared to the saline-control)

Functional Lung Measurements in IT Bleomycin-Induced Pulmonary Fibrosis and Reference Compounds

IT-Bleomycin administered animals have lung mechanics measured on Day 21 using a flexiVent mechanical ventilator. Improved lung function is observed in mice administered intratracheal bleomycin when treated with either pirfenidone or anti-TGFβ neutralizing antibody. (**p<0.01 compared to the naïve group)

Optimal effects of reference compounds in IT Bleomycin-Induced Pulmonary Fibrosis

Lungs from IT-Bleomycin administered animals were collected on Day 21, processed for histopathology, and a modified Ashcroft score was assigned by a board-certified veterinary pathologist. Fixed tissue was also assessed for percent area of fibrotic lung tissue. Pulverized lung samples were assessed hydroxyproline. Lower levels of fibrosis are observed in mice administered intratracheal bleomycin when treated with either pirfenidone or anti-TGFβ neutralizing antibody. (*p<0.05; **p<0.01; ***p<0.001 compared to the naïve group)

Daniel Lichtman

Managing Partner