Autoradiographic study on the regenerative capability of the epithelium lining the center of the cornea after multiple debridements of its peripheral region

  • Sidney Júlio de Faria-e-Sousa
  • Flávia Leão Barbosa
  • Antonio Haddad
Basic Science

Abstract

Background

The epithelium lining the center of the cornea is assumed to lack stem cells.The purpose is to investigate by autoradiography the regenerative capability of the epithelium lining the central region of the rabbit cornea following seven scrapings of its peripheral lining, during several months.

Methods

After marking the center of the cornea with a 6 mm-diameter trephine, the epithelium outside this area was scraped until reaching the corneoscleral zone. This procedure was repeated seven times on the same eye at intervals of 20 days. One day after the last scraping, 3H-thymidine was injected intravitreally and the corneas processed for autoradiography.

Results

At 2 days after injection, the corneal surface was entirely lined by an epithelium made up by two layers of squamous cells, most of them being labeled with the DNA precursor. A multilayered epithelium was visualized at the center with most of its basal cells also labeled. The limbal epithelium had at least two of its layers labeled with the precursor. At 9 days, the multilayered central unscraped epithelium exhibited labeled cells not only in the basal but also in its suprabasal layers. The labeling index (labeled nuclei/100 cells) for its basal stratum was very close to 100%. A similar feature was observed at 16 days, except that the mutilayered central epithelium was seen lining a larger area when compared to the precedent interval and that it exhibited evidences for vertical renewal.

Conclusions

The epithelium lining the central region of the cornea—where it was assumed that stem cells do not exist—exhibited capability for regeneration and self-renewal in spite of seven consecutive debridements of its periphery. No evidence was found for transposition of limbal epithelial cells to the center of the cornea during the early merger of the epithelial sliding fronts.

Keywords

Cornea Epithelium Stem cells Limbus Regeneration 

Notes

Acknowledgements

Gratitude is expressed to Vani M. A. Correa, Maria D. S. Ferreira, José A. Maulin and Domingos S. Souza Fº for their technical assistance. Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brasil) and FAEPA (University Hospital Foundation).

References

  1. 1.
    DePaiva CS, Pflugfelder SC, Li D-Q (2006) Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Stem Cells 24:368–375CrossRefGoogle Scholar
  2. 2.
    Dua HS, Kulkarni B, Franzco RS (2006) Quest for limbal stem cells. Clin Exp Ophthalmol 34:1–2CrossRefGoogle Scholar
  3. 3.
    DePaiva CS, Chen Z, Corrales RM, Pflugfelder SC, Li D-Q (2005) ABCG2 tranporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells 23:63–73CrossRefGoogle Scholar
  4. 4.
    DiIorio E, Barbaro V, Ruzza A, Ponzin D, Pellegrini G, DeLuca M (2005) Isoforms of Np63 and the migration of ocular limbal cells in human corneal regeneration. Proc Natl Acad Sci U S A 102:9523–9528CrossRefGoogle Scholar
  5. 5.
    Dua HS, Shanmuganathan VA, Powell-Richards AO, Tighe PJ, Joseph A (2005) Limbal epithelial crypts: a novel anatomical structure an a putative limbal stem cell niche. Br J Ophthalmol 89:529–532CrossRefPubMedGoogle Scholar
  6. 6.
    Schlötzer-Schrehardt U, Kruse FE (2005) Identification and characterization of limbal stem cells. Exp Eye Res 81:247–264PubMedGoogle Scholar
  7. 7.
    Chen Z, DePaiva CS, Luo L, Kretzer F, Pflugfelder SC, Li D-Q (2004) Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cells 22:355–366CrossRefPubMedGoogle Scholar
  8. 8.
    Zieske JD, Bukusoglu G, Yankauckas MA (1992) Characterization of a potential marker for corneal epithelial stem cells. Invest Ophthalmol Vis Sci 33:143–152PubMedGoogle Scholar
  9. 9.
    Schofield R (1983) The stem cell system. Biomed Pharmacother 37:375–380PubMedGoogle Scholar
  10. 10.
    Goldberg MF, Bron AJ (1982) Limbal palisades of Vogt. Trans Am Ophthalm Soc 80:155–171Google Scholar
  11. 11.
    Davanger M, Evensen A (1971) Role of the pericorneal papillary structure in the renewal of corneal epithelium. Nature 229:560–561CrossRefPubMedGoogle Scholar
  12. 12.
    Chang C-Y, Green CR, McGhee CNJ, Sherwin T (2008) Acute wound healing in the human central corneal epithelium appears to be independent of limbal stem cell influence. Invest Ophthalmol Vis Sci 49:5279–5286CrossRefPubMedGoogle Scholar
  13. 13.
    Majo F, Rochat A, Nicolas M, Jaoudé GA, Barrandon Y (2008) Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 456:250–255CrossRefPubMedGoogle Scholar
  14. 14.
    Chen W, Hara K, Tian Q, Zhao K, Yoshitomi T (2007) Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2. Exp Eye Res 84:626–634CrossRefPubMedGoogle Scholar
  15. 15.
    Li W, Hayashica Y, Chen Y-T, Tseng SCG (2007) Niche regulation of corneal epithelial stem cells at the limbus. Cell Research 17:26–36CrossRefPubMedGoogle Scholar
  16. 16.
    Revoltella RP, Papini S, Rosellini A, Michelini M (2007) Epithelial stem cells of the eye surface. Cell Prolif 40:445–461CrossRefPubMedGoogle Scholar
  17. 17.
    Charukamnoetkanok P (2006) Corneal stem cells: bridging the knowledge gap. Seminars Ophthalmol 21:1–7CrossRefGoogle Scholar
  18. 18.
    Chee KYH, Kicic A, Franzco SJW (2006) Limbal stem cells: the search for a marker. Clin Exp Ophthalmol 34:64–73CrossRefGoogle Scholar
  19. 19.
    Pajoohesh Ganji A, Stepp MA (2005) In search of markers for the stem cells of the corneal epithelium. Biol Cell 97:265–276CrossRefPubMedGoogle Scholar
  20. 20.
    Boulton M, Albon J (2004) Stem cells in the eye. Int J Biochem Cell Biol 36:643–657CrossRefPubMedGoogle Scholar
  21. 21.
    Dua HS, Joseph A, Shanmuganathan VA, Jones RE (2003) Stem cell differentiation and the effects of deficiency. Eye 17:877–885CrossRefPubMedGoogle Scholar
  22. 22.
    Lyngholm M, Vorum H, Nielsen K, Ostergaard M, Honoré B, Ehlers N (2008) Differences in the protein expression in limbal versus central human corneal epithelium – a search for stem cell markers. Exp Eye Res 87:96–105CrossRefPubMedGoogle Scholar
  23. 23.
    Kopriwa BM, Leblond CP (1962) Improvements in the coating technique for radioautography. J Histochem Cytochem 10:269–284Google Scholar
  24. 24.
    Rogers AW (1979) Techniques of autoradiography, 2nd edn. Elsevier, AmsterdamGoogle Scholar
  25. 25.
    Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2008) The cell cycle. In: Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (eds) Molecular Biology of the Cell. 5th Edition. Garland Science, New York, NY, pp 1053–1114Google Scholar
  26. 26.
    Leblond CP (1965) The time dimension in histology. Am J Anat 116:1–28CrossRefPubMedGoogle Scholar
  27. 27.
    Leblond CP (1991) Time dimension in cell biology: a radioautographic survey of dynamic features of cells, cell components, and extracellular matrix. Protoplasma 160:5–38CrossRefGoogle Scholar
  28. 28.
    Hanna C, O’Brien JE (1960) Cell production and migration in the epithelial layer of the cornea. Arch Ophthalmol 64:536–541PubMedGoogle Scholar
  29. 29.
    Lavker RM, Tseng SCG, Sun TT (2004) Corneal epithelial stem cell at the limbus: looking at some old problems from a new angle. Exp Eye Res 78:433–446CrossRefPubMedGoogle Scholar
  30. 30.
    Lehrer MS, Sun T-T, Lavker RM (1998) Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation. J Cell Sci 111:2867–2875PubMedGoogle Scholar
  31. 31.
    Bron AJ, Tripathi RC, Tripathi BJ (1997) Wolff’s anatomy of the eye and orbit, 8th edn. Chapman & Hall Medical, London, UK, pp 233–279Google Scholar
  32. 32.
    Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM (1989) Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell 57:201–209CrossRefPubMedGoogle Scholar
  33. 33.
    Barbosa FL, Faria-e-Sousa SJ, Góes RM, Haddad A (2009) Regeneration of the corneal epithelium after debridement of its central region: an autoradiographic study on rabbits. Curr Eye Res 34:636–645CrossRefPubMedGoogle Scholar
  34. 34.
    Góes RM, Barbosa FL, Faria-e-Sousa SJ, Haddad A (2008) Morphological and autoradiographic studies on the corneal and limbal epithelium of rabbits. Anat Rec 291:191–203CrossRefGoogle Scholar
  35. 35.
    Haddad A (2000) Renewal of the rabbit corneal epithelium as investigated by autoradiography after intravitreal injection of 3H-thymidine. Cornea 19:378–383CrossRefPubMedGoogle Scholar
  36. 36.
    Beebe DC, Masters BR (1996) Cell lineage and the differentiation of corneal epithelium cells. Invest Ophthalmol Vis Sci 37:1815–1825PubMedGoogle Scholar
  37. 37.
    Haskjold E, Bjerknes R, Bjerknes E (1989) Migration of cells in the rat corneal epithelium. Acta Ophthalmol 67:91–96Google Scholar
  38. 38.
    Huang AJW, Tseng SCG (1991) Corneal epithelial wound healing in the absence of limbal epithelium. Invest Ophthalmol Vis Sci 32:96–105PubMedGoogle Scholar
  39. 39.
    Kruse FE, Chen JJY, Tsai RJF, Tseng SCG (1990) Conjunctival transdifferentiation is due to the incomplete removal of limbal basal epithelium. Invest Ophthalmol Vis Sci 31:1903–1913PubMedGoogle Scholar
  40. 40.
    Schwab IR, Isseroff RR (2000) Bioengineered corneas – the promise and the challenge. New Engl J Med 343:136–138CrossRefPubMedGoogle Scholar
  41. 41.
    Tsai RJF, Li LM, Chen JK (2000) Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. New Engl J Med 343:86–93CrossRefPubMedGoogle Scholar
  42. 42.
    Barbaro V, Testa A, DiIorio E, Mavilio F, Pellegrini G, DeLuca M (2007) C/EBP regulates cell cycle and self-renewal of human limbal stem cells. J Cell Biol 177:1037–1049CrossRefPubMedGoogle Scholar
  43. 43.
    Salpeter MM, Budd GG, Mattimoe S (1974) Resolution in autoradiography using semithin sections. J Histochem Cytochem 22:217–222PubMedGoogle Scholar
  44. 44.
    Schantz A, Schecter A (1965) Iron-hematoxilin and safrarin O as a polychrome stain for Epon sections. Stain Technol 40:279–282PubMedGoogle Scholar
  45. 45.
    Hutcheon AEK, Sippel KC, Zieske JD (2007) Examination of the regeneration of epithelial barrier function following superficial keratectomy. Exp Eye Res 84:32–38CrossRefPubMedGoogle Scholar
  46. 46.
    Kuwabara T, Perkins DG, Cogan DG (1976) Sliding of the epithelium in experimental corneal wounds. Invest Ophthalmol 15:4–14PubMedGoogle Scholar
  47. 47.
    Thoft RA, Friend J (1983) The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 24:1442–1443PubMedGoogle Scholar
  48. 48.
    Szerenyi K, Wang XW, Gabrielian K, LaBree L, McDonnell PJ (1994) Immunohistochemistry with 5-bromo-2-deoxyuridine for visualization of mitotic cells in the corneal epithelium. Cornea 13:487–492CrossRefPubMedGoogle Scholar
  49. 49.
    Nagasaki T, Zhao J (2003) Centripetal movement of corneal epithelial cells in the normal adult mouse. Invest Ophthalmol Vis Sci 44:558–566CrossRefPubMedGoogle Scholar
  50. 50.
    Buck RC (1985) Measurements of centripetal migration of normal corneal epithelial cells in the mouse. Invest Ophthalmol Vis Sci 26:1296–1299PubMedGoogle Scholar
  51. 51.
    Chahud F, Ramalho LNZ, Ramalho FS, Haddad A, Roque-Barreira MC (2009) The lectin KM+ induces corneal epithelial wound healing in rabbits. Int J Exp Pathol 90:166–173CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Sidney Júlio de Faria-e-Sousa
    • 1
  • Flávia Leão Barbosa
    • 1
  • Antonio Haddad
    • 2
  1. 1.Departamento de OftalmologiaFaculdade de Medicina de Ribeirão Preto/USPRibeirão PretoBrasil
  2. 2.Departamento de Biologia CelularFaculdade de Medicina de Ribeirão Preto/USPRibeirão PretoBrasil

Personalised recommendations