Korean J Ophthalmol.  2015 Oct;29(5):344-350. 10.3341/kjo.2015.29.5.344.

Comparison of Cytotoxic Effects on Rabbit Corneal Endothelium between Preservative-free and Preservative-containing Dorzolamide/timolol

• Kwon J
• Heo JH
• Kim HM
• Song JS

• KMID: 2363809
• DOI: http://doi.org/10.3341/kjo.2015.29.5.344

Abstract

PURPOSE
To evaluate and compare the toxic effects of eyedrops containing a fixed combination of 2.0% dorzolamide and 0.5% maleate timolol with or without preservatives on rabbit corneal endothelium.
METHODS
This study was performed with 22 eyes of New Zealand white rabbits. Dorzolamide/timolol eyedrops with preservative (Cosopt group) or without preservative (Cosopt-S group) were diluted with a balanced salt solution at a 1 : 1 ratio. We injected 0.1 mL of diluted Cosopt into the anterior chamber of left eyes and an equal volume of diluted Cosopt-S into the anterior chamber of right eyes. Corneal thickness, corneal haze, and conjunctival injection were measured before and 24 hours after treatment. Endothelial damage was compared between both eyes by vital staining (alizarin red/trypan blue staining), live/dead cell assay, TUNEL assay, and scanning electron microscopy.
RESULTS
Corneal endothelial damage was severe in the Cosopt group. Cosopt-treated eyes exhibited remarkable corneal edema and prominent apoptosis of endothelial cells. In addition, the live/dead cell assay revealed many dead cells in the endothelium, and scanning electron microscopy analysis showed that corneal endothelial cells exhibited a partial loss of microvilli on the surface as well as extensive destruction of intercellular junctions. However, in the Cosopt-S group, corneal edema was mild and the damage to the corneal endothelium was minimal.
CONCLUSIONS
The main cause of corneal endothelial toxicity was due to the preservative in the dorzolamide/timolol fixed combination eyedrops, and not the active ingredient. Thus, it appears to be safer to use preservative-free eyedrops during the early postoperative period.

Keyword

Cytotoxicity; Endothelium; Ophthalmic solutions; Rabbits

MeSH Terms

Animals
Anterior Chamber/drug effects
Apoptosis
Corneal Edema/chemically induced/*pathology
Disease Models, Animal
Drug Combinations
Endothelium, Corneal/drug effects/*pathology
In Situ Nick-End Labeling
Ophthalmic Solutions
Rabbits
Sulfonamides/administration & dosage/*toxicity
Thiophenes/administration & dosage/*toxicity
Timolol/administration & dosage/*toxicity
Drug Combinations
Ophthalmic Solutions
Sulfonamides
Thiophenes
Timolol

Figure

• 

  Fig. 1 (A) Slit lamp photograph of an eye 24 hours after injection of Cosopt. A rabbit eye in the Cosopt group showing severe corneal haze and conjunctival vascular injection. (B) Slit lamp photograph of an eye 24 hours after injection of Cosopt-S. A rabbit eye in the Cosopt-S group showing minimal corneal haze and conjunctival vascular injection, the extent of which was much more mild than that of Cosopt-treated eyes. (C) Histopathologic photomicrograph of a rabbit cornea 24 hours after injection of Cosopt. Cornea showing severe stromal edema. Many endothelial cells were lost (inset). (D) Histopathologic photomicrograph of a rabbit cornea 24 hours after injection of Cosopt-S. Significant stromal edema is absent. A single layer of endothelium is well observed (inset) (hematoxylin and eosin, ×40; inset ×400).

• 

  Fig. 2 (A) Vital staining of corneal endothelium with trypan blue and alizarin red 24 hours after Cosopt injection. Extensive endothelial cell damage is noted, resulting in nuclei stained with trypan blue. The corneal endothelial cells are enlarged and have lost their normal hexagonal pattern (×400). (B) Vital staining of corneal endothelium with trypan blue and alizarin red 24 hours after injection of Cosopt-S. The corneal endothelial cells exhibit a normal hexagonal pattern, and some enlarged endothelial cells can be observed (×400). (C) Live/dead cell assay on corneal endothelium 24 hours after injection of Cosopt. Many endothelial cells are dead as evidenced by red-stained nuclei (×200). (D) Live/dead cell assay on corneal endothelium 24 hours after injection of Cosopt-S. Few dead cells are present (×200).

• 

  Fig. 3 (A) Comparison of the number of dead cells from live/dead cell assay and TUNEL(+) cells in 5 consecutive microscopic fields between the Cosopt and Cosopt-S groups (×400). (B) TUNEL stain of rabbit cornea 24 hours after Cosopt injection. Several TUNEL(+) cells are present in the endothelial cell layer. (C) TUNEL stain of rabbit cornea 24 hours after Cosopt-S injection. TUNEL-positive cells are absent (×400).

• 

  Fig. 4 (A) Photograph of scanning electron microscopy 24 hours after Cosopt injection. Corneal endothelial cells have lost microvilli on the cell surface and intercellular junctions are extensively destroyed (×500). (B) Photograph of scanning electron microscopy 24 hours after Cosopt-S injection. Corneal endothelial cells continue to exhibit a hexagonal appearance with distinct microvilli on the cell surface (×500). SE = secondary electron; U = upper detector.

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