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Time-Dependent Resolution of Collagen Deposition During Skin Repair in Rats: A Correlative Morphological and Biochemical Study

Published online by Cambridge University Press:  05 November 2015

Rômulo D. Novaes ,
Marli C. Cupertino ,
Mariaurea M. Sarandy ,
André Souza ,
Evelise A. Soares  and
Reggiani V. Gonçalves
Rômulo D. Novaes*
Affiliation:
Department of Structural Biology, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 700, Campus Universitário, Centro, Alfenas, MG 37130-000, Brazil
Marli C. Cupertino
Affiliation:
Department of General Biology, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil
Mariaurea M. Sarandy
Affiliation:
Department of General Biology, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil
André Souza
Affiliation:
Department of Animal Biology, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil
Evelise A. Soares
Affiliation:
Department of Anatomy, Federal University of Alfenas, Alfenas, MG 37130-000, Brazil
Reggiani V. Gonçalves
Affiliation:
Department of Animal Biology, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil
*
*Corresponding author. [email protected]
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Abstract

Skin samples were used to compare microscopy methods used to quantify collagen with potential applicability to resolve time-dependent collagen deposition during skin wound healing in rats. Skin wounds by secondary intention were made in rats and tissue fragments were collected every 7 days for 21 days. Collagen content determined by biochemical analysis was compared with collagen measured by point counting (PC) on histological skin sections stained by Gomori’s trichrome method (Trichrome/PC), Sirius red under polarized light (PL) microscopy (Sirius red/PL-PC), and computational color segmentation (CS) applied to sections stained with Sirius red (Sirius red/PL-CS). All microscopy methods investigated resolved the time-dependent dynamics of collagen deposition in scar tissue during skin wound healing in rats. Collagen content measured by Sirius red/PL-PC and Sirius red/PL-CS was significantly lower when compared with Trichrome/PC. The Trichrome/PC method provided overestimated values of collagen compared with biochemical analysis. In the early stages of wound healing, which shows high production of noncollagenous molecules, Sirius red/PL-CS and Sirius red/PL-PC methods were more suitable for quantification of collagen fibers. Trichrome staining did not allow clear separation between collagenous and noncollagenous elements in skin samples, introducing a marked bias in collagen quantification.

Keywords

dermisextracellular matrixmorphologypathologywound healing
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 21 , Issue 6 , December 2015 , pp. 1482 - 1490
Copyright
© Microscopy Society of America 2015 

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References

Amadeu, T.P. & Costa, A.M. (2006). Nitric oxide synthesis inhibition alters rat cutaneous wound healing. J Cutan Pathol 33, 465–473.Google Scholar
Ashkani-Esfahani, S., Zarifi, F., Asgari, Q., Samadnejad, A.Z., Rafiee, S. & Noorafshan, A. (2014). Taurine improves the wound healing process in cutaneous leishmaniasis in mice model, based on stereological parameters. Adv Biomed Res 3, 204.Google ScholarPubMed
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 7, 248–254.Google Scholar
Dapson, R.W., Fagan, C., Kiernan, J.A. & Wickersham, T.W. (2011). Certification procedures for sirius red F3B (CI 35780, Direct red 80). Biotech Histochem 86, 133–139.CrossRefGoogle ScholarPubMed
El-Domyati, M., Attia, S., Saleh, F., Brown, D., Birk, D.E., Gasparro, F., Ahmad, H. & Uitto, J. (2002). Intrinsic aging vs. photoaging: A comparative histopathological, immunohistochemical, and ultrastructural study of skin. Exp Dermatol 11, 398–405.Google Scholar
Gomori, G. (1950). A rapid one-step trichrome stain. Am J Clin Pathol 20, 661–664.Google Scholar
Gonçalves, R.V., Novaes, R.D., Cupertino, M.C., Araújo, B.M., Vilela, E.F., Machado, A.T., Leite, J.P. & Matta, S.L. (2014). Bathysa cuspidata extract modulates the morphological reorganization of the scar tissue and accelerates skin wound healing in rats: A time-dependent study. Cells Tissues Organs 199, 266–277.CrossRefGoogle ScholarPubMed
Gonçalves, R.V., Novaes, R.D., Cupertino, M.C., Moraes, B., Leite, J.P., Peluzio, M.C., Pinto, M.V. & da Matta, S.L. (2012). Time-dependent effects of low-level laser therapy on the morphology and oxidative response in the skin wound healing in rats. Lasers Med Sci 28, 383–390.Google Scholar
Gonçalves, R.V., Novaes, R.D., Matta, S.L.P., Benevides, G.P., Faria, F.R. & Pinto, M.V.M. (2010). Comparative study of the effects of gallium-aluminum-arsenide laser photobiomodulation and healing oil on skin wounds in Wistar rats: A histomorphometric study. Photomed Laser Surg 28, 597–602.CrossRefGoogle ScholarPubMed
Grinnell, F. (2003). Fibroblast biology in three-dimensional collagen matrices. Trend Cell Biol 13, 264–269.Google Scholar
Hodde, J. (2002). Naturally occurring scaffolds for soft tissue repair and regeneration. Tissue Eng 8, 295–308.Google Scholar
Junqueira, L.C.U., Bignolas, G. & Brentani, R.R. (1979). Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11, 447–455.CrossRefGoogle ScholarPubMed
Kiernan, J.A. (2008). Methods for connective tissue. In Histological and Histochemical Methods. Theory and Practice, Kiernan J.A. (Ed.), pp. 190–213. London: Arnold.Google Scholar
Lattouf, R., Younes, R., Lutomski, D., Naaman, N., Godeau, G., Senni, K. & Changotade, S. (2014). Picrosirius red staining: A useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem 62, 751–758.Google Scholar
Lucariello, A., Cinelli, M., De Novellis, A., Nikolopoulos, C., Esposito, V. & Guerra, G. (2014). Effects of the combined use of a dermal substitute with a cleansing process in the simulation of autologous skin: A pilot study. In Vivo 28, 639–643.Google Scholar
Mandarim-de-Lacerda, C.A., Fernandes-Santos, C. & Aguila, M.B. (2010). Image analysis and quantitative morphology. In Histology Protocols: Methods in Molecular Biology, Hewitson T.D. & Darby J.A. (Eds.), pp. 211–225. New Jersey: Humana Press.Google Scholar
Martinello, T., Pascoli, F., Caporale, G., Perazzi, A., Iacopetti, I. & Patruno, M. (2015). Might the Masson trichrome stain be considered a useful method for categorizing experimental tendon lesions? Histol Histopathol 30, 963–969.Google Scholar
Moraes, G.H.K., Rodrigues, A.C.P., Silva, F.A., Rostagno, H.S., Minafra, C.S. & Bigonha, S.M. (2010). Effects of dietary L-glutamic acid and K vitamin in the biochemical composition in femurs of broilers at 14 days of age. R Bras Zootec 39, 796–800.Google Scholar
Novaes, R.D., Gonçalves, R.V., Cupertino, M.C., Araújo, B.M., Rezende, R.M., Santos, E.C., Leite, J.P. & Matta, S.L. (2014). The energy density of laser light differentially modulates the skin morphological reorganization in a murine model of healing by secondary intention. Int J Exp Pathol 95, 138–146.Google Scholar
Novaes, R.D., Penitente, A.R., Gonçalves, R.V., Talvani, A., Peluzio, M.C., Neves, C.A., Natali, A.J. & Maldonado, I.R. (2013). Trypanosoma cruzi infection induces morphological reorganization of the myocardium parenchyma and stroma, and modifies the mechanical properties of atrial and ventricular cardiomyocytes in rats. Cardiovasc Pathol 22, 270–279.Google Scholar
Novak, K., Polzer, S., Tichy, M. & Bursa, J. (2015). Automatic evaluation of collagen fiber directions from polarized light microscopy images. Microsc Microanal 21, 863–875.Google Scholar
Pierce, G.F., Vande Berg, J., Rudolph, R., Tarpley, J. & Mustoe, T.A. (1991). Platelet-derived growth factor-BB and transforming growth factor beta 1 selectively modulate glycosaminoglycans, collagen, and myofibroblasts in excisional wounds. Am J Pathol 138, 629–646.Google Scholar
Prabhu, V., Rao, S.B., Chandra, S., Kumar, P., Rao, L., Guddattu, V., Satyamoorthy, K. & Mahato, K.K. (2012). Spectroscopic and histological evaluation of wound healing progression following low level laser therapy (LLLT). J Biophotonics 5, 168–184.Google Scholar
Prabhu, V., Rao, S.B., Fernandes, E.M., Rao, A.C., Prasad, K. & Mahato, K.K. (2014). Objective assessment of endogenous collagen in vivo during tissue repair by laser induced fluorescence. PLoS One 9, e98609.Google Scholar
Rich, L. & Whittaker, P. (2005). Collagen and picrosirius red staining: A polarized light assessment of fibrillar hue and spatial distribution. Braz J Morphol Sci 22, 97–104.Google Scholar
Sarandy, M.M., Novaes, R.D., Matta, S.L.P., Mezencio, J.M.S., Silva, M.B., Zanuncio, J.C. & Goncalves, R.V. (2015). Ointment of Brassica oleracea var. capitata matures the extracellular matrix in skin wounds of Wistar rats. Evid Based Complement Altern Med 2015, 1–9, ID 919342.Google Scholar
Seifert, A.W. & Maden, M. (2014). New insights into vertebrate skin regeneration. Int Rev Cell Mol Biol 310, 129–169.Google Scholar
Tasanen, K., Hämäläinen, E.R., Palatsi, R. & Oikarinen, A. (1996). Quantification of Pro alpha 1(I) collagen mRNA in skin biopsy specimens: Levels of transcription in normal skin and in granuloma annulare. J Invest Dermatol 107, 314–317.Google Scholar
Weibel, E.R. (1979). Stereological Methods. Practical Methods for Biological Morphometry. London: Academic Press.Google Scholar
Werner, S. & Grose, R. (2003). Regulation of wound healing by growth factors and cytokines. Physiol Rev 83, 835–870.Google Scholar
Wong, V.W., Longaker, M.T. & Gurtner, G.C. (2012). Soft tissue mechanotransduction in wound healing and fibrosis. Semin Cell Dev Biol 23, 981–986.Google Scholar