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

Our plain-English summary of A novel method for accurate collagen and biochemical assessment of pulmonary tissue utilizing one animal, by C.R. Kliment, J.M. Englert, L.P. Crum, et al (International Journal of Clinical and Experimental Pathology, 2011).

The study

• Authors: C.R. Kliment, J.M. Englert, L.P. Crum, T.D. Oury
• Journal: International Journal of Clinical and Experimental Pathology
• Published: 2011;4(4):349-55
• Cited by: 84 articles, as counted by NIH iCite
• Read it: PubMed · Free full text

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.

In plain English

This was a laboratory methods study in mice, not people. The authors sought to measure lung scarring (collagen) and run other protein tests using one animal instead of two. Eight-week-old male mice received a single dose of 0.14 mg crocidolite asbestos or 0.14 mg titanium dioxide (an inert control particle) into the windpipe, and lungs were collected 14 days later. For five mice per treatment both lungs were dried whole. For another five, both lungs were homogenized and half the liquid was tested. Using the standard hydroxyproline test, both approaches showed a greater than three-fold increase in asbestos-treated mice compared with controls, and total collagen did not differ between approaches. A second test, the Sirius red dye assay, showed a 25% increase compared with 300% by hydroxyproline, and only a variable trend for newer, salt-soluble collagen. The authors concluded their method measures collagen accurately from a single mouse, while the Sirius red assay tends to overestimate collagen amounts and underestimate the change. They noted it requires careful homogenization in identical volumes.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment comparing laboratory methods for measuring lung collagen (fibrosis)
• Who was studied: Wild-type C57BL/6 male mice, 8 weeks old, randomized into two groups of 10. Each treatment was split into two processing groups of 5 mice. The study was done at the University of Pittsburgh.
• Exposure: Single intratracheal injection of 0.14 mg crocidolite asbestos, or 0.14 mg titanium dioxide as an inert control particle. Lungs were collected on day 14.
• Main results:
  • With the hydroxyproline assay, both processing methods (whole dried lungs and half of a whole-lung homogenate) showed a greater than three-fold increase in lung hydroxyproline in asbestos-treated mice compared with titanium dioxide controls (p less than 0.05).
  • Total collagen per total lung did not differ between the two tissue processing methods.
  • The Sirius red assay showed a significant increase in collagen in asbestos-treated lung samples containing both insoluble and soluble collagens.
  • The increase between control and asbestos-treated lungs was 25% by the Sirius red assay, compared with 300% by the hydroxyproline assay.
  • For salt-soluble collagen only, the Sirius red assay detected a trend toward an increase, with much variability.
  • Sirius red values for total collagen (insoluble plus soluble) were very similar to values for soluble collagen alone, when the former should have been much greater.
• The authors' conclusion: The authors concluded that their method allows collagen assessment and biochemical analysis from a single mouse, with accurate data and fewer animals. They regarded the Sirius red assay as complementary for relative changes, but said it may overestimate collagen and may not reflect changes in fibrosis seen with the hydroxyproline assay.
• Limitations the authors state:
  • The new procedure requires careful homogenization of tissues in identical volumes to avoid introducing error.
  • The Sirius red assay tests a small fraction of the sample, so small staining variations are amplified in the calculations.
  • The Sirius red assay may not reflect significant changes in fibrosis; the authors suggested the dye may bind non-collagen proteins.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Asbestosis, Crocidolite.

Medical terms in this study, explained in our Lexicon: Collagen, Crocidolite.

Related studies

• A protective role for T lymphocytes in asbestos-induced pulmonary inflammation and collagen deposition — E. Corsini, M.I. Luster, et al. (1994)
• Collagen synthesis and degradation during the development of asbestos-induced pulmonary fibrosis — M.G. Arden, I.Y. Adamson (1992)
• Collagen accumulation over time in the murine lung after exposure to crocidolite asbestos or Libby amphibole — A.M. Smartt, M. Brezinski, et al. (2010)
• Epithelial-mesenchymal transition involved in pulmonary fibrosis induced by multi-walled carbon nanotubes via TGF-beta/Smad signaling pathway — T. Chen, H. Nie, et al. (2014)
• Paradoxical function for the receptor for advanced glycation end products in mouse models of pulmonary fibrosis — J.M. Englert, C.R. Kliment, et al. (2011)
• Pulmonary response of mice to fiberglass: cytokinetic and biochemical studies — I.Y. Adamson, H. Prieditis, et al. (1995)

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