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Int J Clin Exp Pathol 2011;4(4):349-355

Original Ariticle
A novel method for accurate collagen and biochemical assessment of pulmonary tissue
utilizing one animal

Corrine R. Kliment, Judson M. Englert, Lauren P. Crum, Tim D. Oury

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA USA.

Received April 3, 2011; accepted April 10; Epub April 12, 2011; published April 30, 2011

Abstract: AIM: The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while
decreasing animal use. METHODS:  8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or
control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis.
Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate
assay. RESULTS: The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical
results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay
showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of
collagen and underestimated changes between control and fibrotic lungs. CONCLUSIONS: The proposed method provides accurate
quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The
Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values
tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.  

Keywords: Pulmonary fibrosis, hydroxyproline, Sirius red, asbestos, collagen quantification

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Address all correspondence to:
Dr. Tim D. Oury, MD, PhD
Department of Pathology
University of Pittsburgh School of Medicine
Phone: 412-624-8888
E-mail: tdoury@pitt.edu