Refined Murine Model of Idiopathic Pulmonary Fibrosis

Pulmonary fibrosis is a devastating and irreversible disease characterized by fibrotic scars and bronchialization of alveoli. This research addresses the limitations in existing animal models by introducing an improved method in mice that more closely mimics the pathology of pulmonary fibrosis. The standard model of pulmonary fibrosis uses intertrachial injection of a single dose of bradiomisine, but fails to mimic the key features. A method using fault A repetitive injections is developed, but it is time consuming and results in high mortality. We use a twice repeated oropharyngeal injection to induce pulmonary fibrosis. It recapitulates the core traits of the disease while saving time and reducing mortality of mice compared to the fault A repetitive injection models.

[Narrator] To begin, weigh each mouse prior to oropharyngeal administration to calculate the required volume of bleomycin solution based on its body weight. Place the mouse on the tracheal injection stand and secure it by hooking its teeth. Using forceps, gently extend the tongue and hold it with the thumb and index finger. Cover the mouse's nose with the middle finger to ensure the solution is delivered properly. Using a 200 microliter pipette tip aspirate the required volume of diluted bleomycin solution and slowly administer the solution into the oropharynx avoiding deep insertion of the pipette tip. Remove the mouse from the stand and place it on a heating pad for recovery. After euthanizing the mice, perform a midline sternotomy to open the thoracic cavity. Perfuse the lungs via the right ventricle using 10 milliliters of ice cold PBS to flush out the blood cells. Insert a cannula into the trachea and inflate the lungs using 1% paraformaldehyde to ensure complete tissue fixation. Excise the lungs and immerse them in 1% paraformaldehyde at room temperature for one hour to allow fixation. For tissue processing, rinse the fixed tissues three times in PBS for five minutes each. Dehydrate the samples using 70% ethanol, ensuring complete dehydration. After dehydration, clear the tissues in xylene for one hour at room temperature. Then embed the tissues in molten paraffin wax at 60 degrees Celsius for at least two hours or until fully infiltrated. Using a rotary microtome, section the embedded tissues into seven micrometer slices. For hematoxylin and eosin staining, perform sequential xylene deparaffinization twice for five minutes. Then rehydrate the slides in 100% ethanol for five minutes. Rehydrate again in 95% ethanol for one minute and 80% ethanol for one minute. Rinse with distilled water. Stain the tissue with hematoxylin for five minutes. Rinse three times with tap water and soak in water for five minutes. Then dip in 80% ethanol for one minute and counterstain with eosin for 45 seconds. After performing dehydration with graded ethanol, clear the slides in xylene by dipping for five minutes twice. Then mount the samples using neutral balsam. For massen staining, after de-waxing, rinse the slides in double distilled water and stain with Ponceau S Fucsion for five seconds. Then rinse with 0.2% weak acid solution for one minute. Differentiate in phosphomolybdic acid solution for five minutes. Then rinse again with weak acid for one minute. Counterstain using Aniline blue for one minute before rinsing again with weak acid. After dehydrating the sections again through graded ethanol, clear the slides in xylene and mount them with resin. For immunohistochemical staining, after de-waxing and rehydrating the paraffin embedded slides perform antigen retrieval by placing the slides in boiling 0.1 molar sodium citrate buffer with 0.5% for 90 seconds. Quench endogenous peroxidase activity with 3% hydrogen peroxide. Permeabilize membranes using 0.5% Trident X 100 in PBS to block nonspecific binding. Add primary antibody to the slides and incubate at four degrees Celsius overnight. Add species matched secondary antibody and incubate again for one hour at room temperature. Now add Streptavidin-HRP and incubate for 30 minutes for immunofluorescence staining after repeating the immunohistochemistry steps. Label biotinylated targets with Streptavidin Alexa Fluor 594 or 647 at a one to 500 dilution for 30 minutes at room temperature. Counterstain nuclei with DAPI at 20 micrograms per milliliter for five minutes and mount the slides using antifade mounting medium. This figure illustrates the histopathological differences in lung fibrosis between single and repetitive bleomycin administration showing hematoxylin and eosin stained sections at 14 days post-injury. Notably, the repetitive bleomycin model exhibited persistent and more extensive fibroblastic foci than the single dose group marked by dense fibrotic clusters. Lung sections from repetitive bleomycin treated mice at 60 days post-injury showed more extensive fibrotic remodeling compared to the single dose group with significant distortion of alveolar structure. Masson's trichrome staining revealed increased collagen deposition in the repetitive model relative to the single dose group. Immunohistochemistry showed elevated expression of alpha smooth muscle actin and hydroxyproline in the repetitive group, indicating persistent myofibroblast activity and matrix accumulation along with a marked increase in keratin five positive epithelial cells. Immunofluorescence revealed Delta N p63 positive basal cell clusters exclusively in the repetitive model absent in single dosed lungs. Additionally, honeycomb like cystic structures were observed in the repetitive model resembling hallmark features of idiopathic pulmonary fibrosis. Bronchialization of alveolar epithelium was also evident in the repetitive injury group.