Collagen pp 169-188 | Cite as

Assessing Collagen Deposition During Aging in Mammalian Tissue and in Caenorhabditis elegans

  • Alina C. Teuscher
  • Cyril Statzer
  • Sophia Pantasis
  • Mattia R. BordoliEmail author
  • Collin Y. Ewald
Part of the Methods in Molecular Biology book series (MIMB, volume 1944)


Proper collagen homeostasis is essential for development and aging of any multicellular organism. During aging, two extreme scenarios are commonly occurring: a local excess in collagen deposition, for instance during fibrosis, or a gradual overall reduction of collagen mass. Here, we describe a histological and a colorimetric method to assess collagen levels in mammalian tissues and in the nematode Caenorhabditis elegans. The first method is the polychrome Herovici staining to distinguish between young and mature collagen ratios. The second method is based on hydroxyproline measurements to estimate collagen protein levels. In addition, we show how to decellularize the multicellular organism C. elegans in order to harvest its cuticle, one of the two major extracellular matrices, mainly composed of collagen. These methods allow assessing collagen deposition during aging either in tissues or in whole organisms.

Key words

Collagen Aging Tissue Herovici staining Hydroxyproline Cuticle Isolation Freeze-cracking Age-synchronizing Extracellular matrix C. elegans 



We thank Marjolein Wildwater for sharing her improved freeze-cracking protocol, Eline Jongsma for her assistance in adapting the freeze-cracking protocol, Salome Brütsch and Hayley Hiebert for their help to develop the C. elegans Herovici staining protocol, Anna Bircher for contributing to the early stages of the cuticle isolation protocols and imaging cuticles, Max Hess for providing the C. elegans example image, and Jan M. Gebauer for bioinformatic prediction of potential prolines in collagens that might become hydroxylated in C. elegans. Some C. elegans strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by the Swiss National Science Foundation [PZ00P3 161512] to S.P. and M.R.B. and [PP00P3 163898] to A.C.T., C.S., and C.Y.E. Alina C. Teuscher and Cyril Statzer contributed equally to this work.


  1. 1.
    Hynes RO (2009) The extracellular matrix: not just pretty fibrils. Science 326:1216–1219CrossRefGoogle Scholar
  2. 2.
    Ricard-Blum S (2011) The collagen family. Cold Spring Harb Perspect Biol 3:a004978–a004978CrossRefGoogle Scholar
  3. 3.
    Sivan S-S, Wachtel E, Tsitron E et al (2008) Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid. J Biol Chem 283:8796–8801CrossRefGoogle Scholar
  4. 4.
    Heinemeier KM, Schjerling P, Heinemeier J et al (2016) Radiocarbon dating reveals minimal collagen turnover in both healthy and osteoarthritic human cartilage. Sci Transl Med 8:346ra90–346ra90CrossRefGoogle Scholar
  5. 5.
    Toyama BH, Hetzer MW (2013) Protein homeostasis: live long, won't prosper. Nat Rev Mol Cell Biol 14:55–61CrossRefGoogle Scholar
  6. 6.
    Kjaer M, Langberg H, Miller BF et al (2005) Metabolic activity and collagen turnover in human tendon in response to physical activity. J Musculoskelet Neuronal Interact 5:41–52PubMedGoogle Scholar
  7. 7.
    Myllyharju J, Kivirikko KI (2001) Collagens and collagen-related diseases. Ann Med 33:7–21CrossRefGoogle Scholar
  8. 8.
    Fisher GJ, Quan T, Purohit T et al (2009) Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin. Am J Pathol 174:101–114CrossRefGoogle Scholar
  9. 9.
    Shoulders MD, Raines RT (2009) Collagen structure and stability. Annu Rev Biochem 78:929–958CrossRefGoogle Scholar
  10. 10.
    Sell DR, Monnier VM (2012) Molecular basis of arterial stiffening: role of glycation–a mini-review. Gerontology 58:227–237CrossRefGoogle Scholar
  11. 11.
    Snedeker JG, Snedeker JG, Gautieri A et al (2014) The role of collagen crosslinks in ageing and diabetes–the good, the bad, and the ugly. Muscles Ligaments Tendons J 4:303–308CrossRefGoogle Scholar
  12. 12.
    Myllyharju J (2004) Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet 20:33–43CrossRefGoogle Scholar
  13. 13.
    Fenske NA, Lober CW (1986) Structural and functional changes of normal aging skin. J Am Acad Dermatol 15:571–585CrossRefGoogle Scholar
  14. 14.
    Shuster S, Black MM, McVitie E (1975) The influence of age and sex on skin thickness, skin collagen and density. Br J Dermatol 93:639–643CrossRefGoogle Scholar
  15. 15.
    López-Otín C, Blasco MA, Partridge L et al (2013) The hallmarks of aging. Cell 153:1194–1217CrossRefGoogle Scholar
  16. 16.
    Franceschi C, Campisi J (2014) Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases. J Gerontol A Biol Sci Med Sci 69(Suppl 1):S4–S9CrossRefGoogle Scholar
  17. 17.
    Wynn TA (2007) Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Investig 117:524–529CrossRefGoogle Scholar
  18. 18.
    Gutiérrez-Fernández A, Soria-Valles C, Osorio FG et al (2015) Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid. EMBO J 34:1875–1888CrossRefGoogle Scholar
  19. 19.
    Flurkey K, Papaconstantinou J, Miller RA et al (2001) Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production. Proc Natl Acad Sci U S A 98:6736–6741CrossRefGoogle Scholar
  20. 20.
    Wilkinson JE, Burmeister L, Brooks SV et al (2012) Rapamycin slows aging in mice. Aging Cell 11:675–682CrossRefGoogle Scholar
  21. 21.
    Ewald CY, Landis JN, Porter Abate J et al (2015) Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity. Nature 519:97–101CrossRefGoogle Scholar
  22. 22.
    Herovici C (1963) A polychrome stain for differentiating precollagen from collagen. Stain Technol 38:204–205Google Scholar
  23. 23.
    McAnulty RJ (2005) Methods for measuring hydroxyproline and estimating in vivo rates of collagen synthesis and degradation. Methods Mol Med 117:189–207PubMedGoogle Scholar
  24. 24.
    Qiu B, Wei F, Sun X et al (2014) Measurement of hydroxyproline in collagen with three different methods. Mol Med Rep 10:1157–1163CrossRefGoogle Scholar
  25. 25.
    Naba A, Clauser KR, Hynes RO (2015) Enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis. J Vis Exp 101:e53057Google Scholar
  26. 26.
    Kramer JM (2005) Basement membranes, WormBook : the online review of C elegans biology. pp 1–15Google Scholar
  27. 27.
    Page AP and Johnstone IL (2007) The cuticle, WormBook : the online review of C elegans biology. pp 1–15Google Scholar
  28. 28.
    Leushner JR, Semple NL, Pasternak J (1979) Isolation and characterization of the cuticle from the free-living nematode Panagrellus silusiae. Biochim Biophys Acta 580:166–174CrossRefGoogle Scholar
  29. 29.
    Cox GN, Kusch M, Edgar RS (1981) Cuticle of Caenorhabditis elegans: its isolation and partial characterization. J Cell Biol 90:7–17CrossRefGoogle Scholar
  30. 30.
    Treuting PM, Snyder JM (2015) Mouse necropsy. Curr Protoc Mouse Biol 5:223–233CrossRefGoogle Scholar
  31. 31.
    Stiernagle T (2006) Maintenance of C. elegans, WormBook : the online review of C elegans biology. pp 1–11Google Scholar
  32. 32.
    Fischer AH, Jacobson KA, Rose J et al (2008) Paraffin embedding tissue samples for sectioning. CSH Protoc 2008:pdb.prot4989PubMedGoogle Scholar
  33. 33.
    Duerr JS (2006) Immunohistochemistry, WormBook : the online review of C elegans biology. pp 1–61Google Scholar
  34. 34.
    Davis BO, Anderson GL, Dusenbery DB (1982) Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans. Biochemistry 21:4089–4095CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Alina C. Teuscher
    • 1
  • Cyril Statzer
    • 1
  • Sophia Pantasis
    • 2
  • Mattia R. Bordoli
    • 2
    Email author
  • Collin Y. Ewald
    • 1
  1. 1.Department of Health Sciences and Technology, Institute of Translational MedicineEidgenössische Technische Hochschule (ETH) ZürichZürichSwitzerland
  2. 2.Department of Biology, Institute of Molecular Health SciencesEidgenössische Technische Hochschule (ETH) ZürichZürichSwitzerland

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