Hostname: page-component-cd9895bd7-hc48f Total loading time: 0 Render date: 2024-12-25T07:05:34.058Z Has data issue: false hasContentIssue false
  • English
  • Français

Methylene Blue Assay for Estimation of Regenerative Re-Epithelialization In Vivo

Published online by Cambridge University Press:  23 February 2017

Maresha Milyavsky  and
Renee Dickie
Maresha Milyavsky
Affiliation:
Department of Biological Sciences, Towson University, Towson, MD 21252, USA
Renee Dickie*
Affiliation:
Department of Biological Sciences, Towson University, Towson, MD 21252, USA
*
*Corresponding author.[email protected]
Get access

Abstract

The rapidity with which epithelial cells cover a wound surface helps determine whether scarring or scar-less healing results. As methylene blue is a vital dye that is absorbed by damaged tissue but not undamaged epidermis, it can be used to assess wound closure. We sought to develop a quantitative methylene blue exclusion assay to estimate the timeframe for re-epithelialization in regenerating appendages in zebrafish and axolotls, two classic model systems of regeneration. Following application of methylene blue to the amputation plane and extensive washing, the regenerating tail was imaged in vivo until staining was no longer visible. The percent area of the amputation plane positive for methylene blue, representing the area of the amputation plane not yet re-epithelialized, was measured for each time point. The loss of methylene blue occurred rapidly, within ~2.5 h in larval and juvenile axolotls and <1 h in adult zebrafish, consistent with high rates of re-epithelialization in these models of regeneration. The assay allows simple, rapid estimation of the time course for regenerative re-epithelialization without affecting subsequent regenerative ability. This technique will permit comparison of re-epithelialization across different strains and stages, as well as under the influence of various pharmacological inhibitors that affect regeneration.

Keywords

regenerationepithelialmethylene blueaxolotlzebrafish
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 23 , Issue 1 , February 2017 , pp. 113 - 121
Copyright
© Microscopy Society of America 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Anghel, E.L., DeFazio, M.V, Barker, J.C., Janis, J.E. & Attinger, C.E. (2016). Current concepts in debridement: Science and strategies. Plast Reconstr Surg 138(3), 82S–93S.CrossRefGoogle ScholarPubMed
Argyris, T. (1976). Kinetics of epidermal production during epidermal regeneration following abrasion in mice. Am J Pathol 83(2), 329–340.Google ScholarPubMed
Atamna, H., Nguyen, A., Schultz, C., Boyle, K., Newberry, J., Kato, H. & Ames, B.N. (2008). Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J 22, 703–712.CrossRefGoogle ScholarPubMed
Block, L., King, T.W. & Gosain, A. (2015). Debridement techniques in pediatric trauma and burn-related wounds. Adv Wound Care 4(10), 596–606.CrossRefGoogle ScholarPubMed
Bruchey, A.K. & Gonzalez-Lima, F. (2008). Behavioral, physiological and biochemical hormetic responses to the autoxidizable dye methylene blue. Am J Pharmacol Toxicol 3(1), 72–79.CrossRefGoogle Scholar
Campbell, L.J. & Crews, C.M. (2008). Wound epidermis formation and function in urodele amphibian limb regeneration. Cell Mol Life Sci 65(1), 73–79.CrossRefGoogle ScholarPubMed
Campbell, L.J., Suárez-Castillo, E.C., Ortiz-Zuazaga, H., Knapp, D., Tanaka, E.M. & Crews, C.M. (2011). Gene expression profile of the egeneration epithelium during axolotl limb regeneration. Dev Dyn 240(7), 1826–1840.CrossRefGoogle Scholar
Carlson, B.M. (2005). Some principles of regeneration in mammalian systems. Anat Rec, B New Anat 287B(1), 4–13.CrossRefGoogle Scholar
Carlson, M.R., Bryant, S.V. & Gardiner, D.M. (1998). Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs. J Exp Zool 282(6), 715–723.3.0.CO;2-F>CrossRefGoogle ScholarPubMed
Celikoz, B., Deveci, M. & Nisanci, A. (1999). Early tangential excision with the guidance of methylene blue application. Ann Burns Fire Disasters 12(4), 217–220.Google Scholar
Chen, Y.W., Lin, J.S., Fong, J.H., Wang, I.K., Chou, S.J., Wu, C.H., Lui, M.T., Chang, C.S. & Kao, S.Y. (2007). Use of methylene blue as a diagnostic aid in early detection of oral cancer and precancerous lesions. Br J Oral Maxillofac Surg 45, 590–591.CrossRefGoogle ScholarPubMed
Chu, M. & Wan, Y. (2009). Sentinel lymph node mapping using near-infrared fluorescent methylene blue. J Biosci Bioeng 107(4), 455–459.CrossRefGoogle ScholarPubMed
Cook, R.P. (1926). The antagonism of acetyl choline by methylene blue. J Physiol 62, 160–165.CrossRefGoogle ScholarPubMed
Cornell, R.S., Meyr, A.J., Steinberg, J.S. & Attinger, C.E. (2010). Débridement of the noninfected wound. J Vasc Surg 52(3), 31S–36S.CrossRefGoogle ScholarPubMed
Davies, M.R.Q., Andendorff, D., RODE, H. & Van Der Riet, R. (1980). Colouring the damaged tissues on the burn wound surface. Burns 6, 156–159.CrossRefGoogle Scholar
Denis, J.-F., Lévesque, M., Tran, S.D., Camarda, A.-J. & Roy, S. (2013). Axolotl as a model to study scarless wound healing in vertebrates: Role of the transforming growth factor beta signaling pathway. Adv Wound Care 2(5), 250–260.CrossRefGoogle Scholar
Donaldson, D.J. & Dunlap, M.K. (1981). Epidermal cell migration during attempted closure of skin wounds in the adult newt: Observations based on cytochalasin treatment and scanning electron microscopy. J Exp Zool 217(1), 33–43.CrossRefGoogle ScholarPubMed
Dorafshar, A.H., Gitman, M., Henry, G., Agarwal, S. & Gottlieb, L.J. (2010). Guided surgical debridement: Staining tissues with methylene blue. J Burn Care Res 31(5), 791–794.CrossRefGoogle ScholarPubMed
Endara, M. & Attinger, C. (2012). Using color to guide debridement. Adv Skin Wound Care 25(12), 549–555.CrossRefGoogle ScholarPubMed
Endo, T., Bryant, S.V. & Gardiner, D.M. (2004). A stepwise model system for limb regeneration. Dev Biol 270(1), 135–145.CrossRefGoogle ScholarPubMed
Ferris, D.R., Satoh, A., Mandefro, B., Cummings, G.M., Gardiner, D.M. & Rugg, E.L. (2010). Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin. Dev., Growth Differ 52(8), 715–724.CrossRefGoogle ScholarPubMed
Genina, E.A., Bashkatov, A.N. & Tuchin, V.V. (2004). Methylene blue diffusion in skin tissue. Proc SPIE 5486, 315–323.Google Scholar
Goss, R.J. (1956). Regenerative inhibition following limb amputation and immediate insertion into the body cavity. Anat Rec 126(1), 15–27.CrossRefGoogle ScholarPubMed
Granick, M., Jacoby, M., Noruthrun, S., Ramazi, O., Datiashvili, R. & Ganchi, P. (2006). Hydrosurgical debridement on chronic wounds. Wounds 18(2), 35–39.Google Scholar
Guimond, J.-C., Lévesque, M., Michaud, P.-L., Berdugo, J., Finnson, K., Philip, A. & Roy, S. (2010). BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs. BMC Dev Biol 10, 15.CrossRefGoogle ScholarPubMed
Kim, W. & Applegate, B.E. (2015). In vivo molecular contrast OCT imaging of methylene blue. Opt Lett 40(7), 1426–1429.CrossRefGoogle ScholarPubMed
Lejoy, A., Arpita, R., Krishna, B. & Venkatesh, N. (2016). Methylene blue as a diagnostic aid in the early detection of potentially malignant and malignant lesions of oral mucosa. Ethiop J Health Sci 26(3), 201–208.Google ScholarPubMed
Lévesque, M., Villiard, E. & Roy, S. (2010). Skin wound healing in axolotls: A scarless process. J Exp Zool B Mol Dev Evol 314(8), 684–697.CrossRefGoogle ScholarPubMed
Mescher, A.L. (1976). Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface. J Exp Zool 195(1), 117–127.CrossRefGoogle ScholarPubMed
Monaghan, J.R., Stier, A.C., Michonneau, F., Smith, M.D., Pasch, B., Maden, M. & Seifert, A.W. (2014). Experimentally induced metamorphosis in axolotls reduces regenerative rate and fidelity. Regeneration (Oxf) 1(1), 2–14.CrossRefGoogle ScholarPubMed
Mu, L., Tang, J., Liu, H., Shen, C., Rong, M., Zhang, Z. & Lai, R. (2014). A potential wound-healing-promoting peptide from salamander skin. FASEB J 28(9), 3919–3929.CrossRefGoogle ScholarPubMed
Murawala, P., Tanaka, E.M. & Currie, J.D. (2012). Regeneration: The ultimate example of wound healing. Semin Cell Dev Biol 23(9), 954–962.CrossRefGoogle ScholarPubMed
Odland, G. & Ross, R. (1968). Human wound repair I. Epidermal regeneration. J Cell Biol 39(1), 9135–9151.CrossRefGoogle ScholarPubMed
Osorio, J.A., Breshears, J.D., Arnaout, O., Simon, N.G., Hastings-Robinson, A.M., Aleshi, P. & Kliot, M. (2015). Ultrasound-guided percutaneous injection of methylene blue to identify nerve pathology and guide surgery. Neurosurg Focus 39(3), E2.CrossRefGoogle ScholarPubMed
Pastar, I., Stojadinovic, O., Yin, N.C., Ramirez, H., Nusbaum, A.G., Sawaya, A., Patel, S.B., Khalid, L., Isseroff, R.R. & Tomic-Canic, M. (2014). Epithelialization in wound healing: A comprehensive review. Adv Wound Care 3(7), 445–464.CrossRefGoogle ScholarPubMed
Quilha, A. & Sire, J.Y. (1999). Spreading, proliferation, and differentiation of the epidermis after wounding a cichlid fish, Hemichromis bimaculatus. Anat Rec 254, 435–451.3.0.CO;2-D>CrossRefGoogle Scholar
Repesh, L.A. & Oberpriller, J.C. (1980). Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens Am J Anat 159(2), 187–208.CrossRefGoogle ScholarPubMed
Richardson, R., Metzger, M., Knyphausen, P., Ramezani, T., Slanchev, K., Kraus, C., Schmelzer, E. & Hammerschmidt, M. (2016). Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals. Development 143, 2077–2088.Google ScholarPubMed
Richardson, R., Slanchev, K., Kraus, C., Knyphausen, P., Eming, S. & Hammerschmidt, M. (2013). Adult zebrafish as a model system for cutaneous wound-healing research. J Invest Dermatol 133(6), 1655–1665.CrossRefGoogle Scholar
Rolfe, K.J. & Grobbelaar, A.O. (2012). A review of fetal scarless healing. ISRN Dermatol 2012, 698034.CrossRefGoogle ScholarPubMed
Satoh, A., Graham, G.M.C., Bryant, S.V. & Gardiner, D.M. (2008). Neurotrophic regulation of epidermal dedifferentiation during wound healing and limb regeneration in the axolotl (Ambystoma mexicanum). Dev Biol 319(2), 321–335.CrossRefGoogle ScholarPubMed
Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7), 671–675.CrossRefGoogle ScholarPubMed
Seifert, A.W., Monaghan, J.R., Voss, S.R. & Maden, M. (2012). Skin regeneration in adult axolotls: A blueprint for scar-free healing in vertebrates. PloS One 7(4), e32875.CrossRefGoogle ScholarPubMed
Shah, M., Foreman, D.M. & Ferguson, M.W. (1995). Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 108(3), 985–1002.CrossRefGoogle ScholarPubMed
Tanaka, E. & Galliot, B. (2009). Triggering the regeneration and tissue repair programs. Development 136(3), 349–353.CrossRefGoogle ScholarPubMed
Tassava, R.A. & Garling, D.J. (1979). Regenerative responses in larval axolotl limbs with skin grafts over the amputation surface. J Exp Zool 208(1), 97–110.CrossRefGoogle ScholarPubMed
Thornton, C.S. (1957). The effect of apical cap removal on limb regeneration in Amblystoma larvae. J Exp Zool 134(2), 357–381.CrossRefGoogle ScholarPubMed
Wallace, H., Watson, A. & Egar, M. (1981). Regeneration of subnormally innervated axolotl arms. J Embryol Exp Morphol 62, 1–11.Google ScholarPubMed
Xiong, Z.-M., Choi, J.Y., Wang, K., Zhang, H., Tariq, Z., Wu, D., Ko, E., LaDana, C., Sesaki, H. & Cao, K. (2016). Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell 15(2), 279–290.CrossRefGoogle ScholarPubMed