Current Issues of Pharmacy and Medical Sciences

Ultrastructural and morphohistochemical study of the influence of benzalkonium chloride on human corneal limbal epithelial cells

Current Issues in Pharmacy and Medical Sciences Vol. 26, No. 4, Pages 461-467


1 Department of Surgery of the Retina and Vitreous, Medical University of Lublin, Lublin, Poland
2 Department of Anatomy, Collegium Medicum, Jagiellonian University of Cracow, Cracow, Poland

DOI: 10.12923/j.2084-980X/26.4/a.25



Within the corneal limbal epithelium there exist limbal stem cells (LSC) which under suitable conditions can regenerate their population or differentiate into corneal epithelial cells. However, upon limbal damage, the cells differentiate irreversibly and do not self-renew. One of the causes of the damage of progenitor cells and their niche is a long-term use of eye drops containing preservatives. While the side effects are related to the antimicrobial activity of such eye drops, damage to cellular and cytoplasmic membranes as well as enzymatic reactions can concurrently cause disorders of normal ocular surface tissue. The aim of this study was to evaluate the toxic effects of the preservative used in eye drops - benzalkonium chloride (BAK) - on human corneal limbal epithelial cells in vitro, and to define the mechanisms of acute limbal cell damage caused by the action of BAK. Ten corneoscleras rims, which were not qualified for transplantation by the Eye Tissue Bank, were obtained from 5 deceased donors aged 39 to 43 years. The tissue fragments (explants) containing corneal limbal epithelial cells were immediately after the explantation subjected to the action of the experimental substance being benzalkonium chloride (BAK) in concentrations of 0.005% and 0.01%. The qualitative analysis of microscopic images of the corneal limbus specimens was performed on tissue sections stained with hematoxylin and eosin using the immunohistochemical method for vimentin and with the use of a transmission electron microscope. The structure of the area of corneal limbus, as well as the morphological characteristics and the ultrastructure of the very limbal cells were evaluated with careful attention to the basal epithelial cells of the limbus. The BAK-treated groups of explants in sections stained by H & E featured characteristics of severe structural disorders of the corneal limbus area. Depletion of the epithelial cells was visible and involved both superficial and deep layers. Immunohistochemical staining for vimentin did not show the expression of this protein. This might have been connected with the damage to the cytoskeleton of limbal epithelial cells and large depletion of cells reaching down to the basement membrane. The images obtained with electron microscopy demonstrate serious defects of cell ultrastructure and, indirectly, abnormal cellular metabolism, including water and electrolyte balance and energy metabolism.This experiment confirmed the significant adverse effect of benzalkonium chloride on the limbal epithelial cells and the possibility of their damage.


limbal stem cell, preservative, benzalkonium chloride, limbal insufficiency


  1. Baudouin C.: Jatrogenne uszkodzenia spojówki i rogówki. In: Choroby twardówki, rogówki i powierzchni oka. Pod redakcją Z. Zagórskiego, G Naumana, P Watsona. Lublin, 2008, wydawn. Czelej
  2. Broadway D., Hitchings R.: Adverse effects of topical antiglaucomatous medications on the conjunctiva. Br J Ophthalmol.,  77: 590-596, 1993.
  3. Burstein N.L.: The effects of topical drugs and preservatives on the tears and corneal epithelium in dry eye. Trans. Ophthalmol. Soc. UK., 104: 402-409, 1985.
  4. Buschke W., Friendenwald J. S., Fleischmann W.: Some factors concerned in the mitotic and wound-healing activities of the corneal epithelium. Trans. Am. Ophthalmol. Soc., 42: 371-83, 1944.
  5. Chibret H.: Conservateurs et pre´parations ophtalmiques: re´alite´s et perspectives. Ann. Pharm. Fr., 55: 228-231, 1997.
  6. Davanger, M., Evensen, A.: Role of the pericorneal papillary structure in renewal of corneal epithelium. Nature.,  229: 560-561, 1971.
  7. De Saint Jean M. et al.: Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells. Invest. Ophthalmol. Vis. Sci., 40: 619-30, 1997.
  8. Deutschle T. et al.: In vitro genotoxicity and cytotoxicity of benzalkonium chloride. Toxicol. in Vitro., 20: 1472-1477, 2006.
  9. Dutot M. et al.: Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis. Invest. Ophthalmol. Vis. Sci.,  47: 2812-9, 2006.
  10. Furrer P., Mayer J.M., Gurny R.: Ocular tolerance of preservatives and alternatives. European Journal of Pharmaceutics and Biopharmaceutics., 53: 263-280, 2002.
  11. Harminder S. D., Azuara-Blanco A.: Limbal Stem Cells of the corneal epithelium. Surv. Ophthalmol., 44: 415-425, 2000.
  12. Kasper M. et al.: Patterns of cytokeratin and vimentin expression in the human eye. Histochemistry., 89: 369-377, 1988.
  13. Kuppens E.V.M.J. et al.: Effect of timolol with and without preservative on the basal tear turnover in glaucoma. Br. J. Ophthalmol., 79: 339-342, 1995.
  14. Lavker R.M. et al.: Relative proliferative rates of limbal and corneal epithelia. Implications of corneal epithelial migration, circadian rhythm, and suprabasally located DNA-synthesizing keratinocytes. Invest. Ophthalmol. Vis. Sci., 32: 1864-1875, 1991.
  15. Lemp M.A., Zimmerman L.E.: Toxic endothelial degeneration in ocular surface disease treated with topical medications containing benzalkonium chloride. Am. J. Ophthalmol., 105: 670-673, 1988.
  16. Li W. et al.: Niche regulation of corneal epithelial stem cells at the limbus. Cell Res., 17: 26-36, 2007.
  17. Seung-Heon Cha, et al.: Corneal epithelial cellular dysfunction from benzalkonium chloride (BAC) in vitro. Clinical and Experimental Ophthalmology., 32: 180-184, 2007.
  18. Shanmuganathan V.A. et al.: Morphological characteristics of the limbal epithelial crypt. Br. J. Ophthalmol., 91: 514-519, 2007.
  19. Sharma A., Coles W.H.: Kinetics of corneal epithelial maintenance and graft loss. A population balance model. Invest. Ophthalmol. Vis. Sci., 30 1962-71, 1989.
  20. Wilson L.A.: To preserve or not to preserve, is that the question? Br. J. Ophthalmol., 80: 583-584, 1996.


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