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Instructions,
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Airway fibrosis in a mouse model of airway
inflammation. Pulmonary fibrosis is defined by excess collagen in the lungs as occurs in human interstitial fibrosis and bronchiolitis obliterans. The excess collagen is found primarily in the centri-acinar and alveolar regions below the epithelium. Excess collagen may result in the airway remodeling thought to be a part of asthma. This experiment utilizes a mouse model in which exposure to ovalbumin results in changes characteristic of asthma including eosinophilic bronchitis, airway hyperresponsiveness and thickening of the sub-epithelial matrix. The authors of this study note that the fibrosis of the airway region in ovalbumin aerosol exposed mice is similar to toxicant-induced fibrosis of the distal lung. These toxicants include bleomycin, paraquat, and oxidant gases. Thus fibrosis can be induced in both the airway region and the peripheral region of the lung. The authors hypothesized that the mechanisms leading to injury and excess collagen in these different regions of the lung might be similar as well. The purpose of this experiment was to determine if there is an increase in collagen biosynthesis and deposition in the subepithelial compartment of mice exposed to ovalbumin. PCR analysis was used to assess mRNA types in fibrotic airways. In addition any relationship of structural changes in the ovalbumin-treated mice to the changes in collagen biosynthesis were examined. Finally B-aminopropionitrile (BAPN) and relaxin, found to be anti-fibrotic in other animal models, were tested for their ability to reduce structural changes in this mouse model of fibrosis. Mice were studied at intervals up to 8 wks after exposure. Materials and methods: Lung isolation and fixation: At the appropriate sacrifice time, mice were killed and the lungs lavaged. Lungs were then either fixed for light microscopy or prepared for analysis of airway 4-hydroxyproline content. Tissue staining and evaluation of fibrosis: Paraffin sections were stained with Picro-Sirius red stain and picric acid for collagen. A 0 – 4 grading system was used to determine degree and depth of stain. Preparation of airway homogenates for assay and assays: Airways were dissected out for the assay of hydroxyproline and total protein. Protein was measured with the Micro BCA Protein Assay Reagent Kit. The colorimetric method of Woessner was used for hydroxyproline assay. RNA was isolated from microdissected airways and reverse transcribed into cDNA. Real-time PCR analysis (RT-PCR) was used to measure different gene products. Airway reactivity: Whole body plethysmography was used to measure hyperresponsiveness of airways to methacholine. An index called "enhanced pause" or Penh was used to compare treatment groups. Results and Discussion: To summarize, within the first 2 weeks of exposure to ovalbumin, inflammation and remodeling of the airways were observed in ovalbumin-sensitized mice. Simultaneously increased airway hyperreactivity was measured and presumed to be related to the remodeling observed. At 4 weeks of exposure, airways had increased collagen and expression of mRNA for types I and III collagen as well as fibrosis. The authors conclude that most of the new collagen in the conducting airways was type 1. This conclusion is drawn from the results of RT-PCR. Relaxin treatment helped to reverse this fibrosis. The authors describe the inflammatory changes in detail noting that the changes they observe seem to support the previously described pathophysiological process of fibrosis development. Airway inflammation, mucous cell hypertrophy and hyperplasia, and modeling of the airway with submucosal fibrosis occurred over time. This process leading to fibrosis is thought to involve lymphocyte and eosinophil recruitment to the lung. TGF-ß is released by the eosinophils and causes differentiation of submucosal fibroblasts in the airways to myofibroblasts. These myofibroblasts are thought to produce the collagen resulting in airway fibrosis. The new collagen was determined to be Type I by PCR analysis of mRNA for the chains in collagens I and III in in conducting airways at 6 wks post-exposure. They also note that airway fibrosis appears to be self-limiting in this model. Collagen is synthesized mainly during the first 2 months of exposure. The question of a correlation between the histological changes during asthma development with functional physiological changes is answered in this experiment. There was an increase in airway hyperreactivity or airway resistance while the fibrosis observed was developing. This correlation was determined with the measurement of Penh, a value considered to be an accurate measurement of airway resistance. Penh was measured after methacholine challenge. The functional changes were found as early as 4 weeks after exposure, which was also the first time an increase in collagen was observed. Currently there is no way to intervene in the pathophysiology of human asthma until late in disease development. This ovalbumin-treated mouse model may allow testing of new drugs for use in humans with asthma. The possibility of therapeutic intervention before the appearance of advanced human asthma is discussed based upon the success of relaxin in preventing collagen accumulation in these ovalbumin treated mouse lungs. Relaxin is an activator of matrix metalloproteinases and inhibits collagen deposition in others models of fibrosis including kidney fibrosis.
Editorial Note: This study does not specify the sex of the BALB/c mouse that was used. I may not have noticed this had I not been reviewing a paper on gender differences in the response of mice to naphthalene (Gender differences in naphthalene metabolism and acute toxicity in lungs). I would assume male mice were used because male animals have traditionally been used in studies. -By: Susan G. Shami, ScD 18 May 03 |