J Korean Ophthalmol Soc.  2015 Dec;56(12):1945-1952. 10.3341/jkos.2015.56.12.1945.

Long-Term Effect of Preservative-Free Sodium Hyaluronate Eye Drop on Human Corneal Epithelial Cell

• Lee JS
• Park JS
• Kim HY

• KMID: 2148759
• DOI: http://doi.org/10.3341/jkos.2015.56.12.1945

Abstract

PURPOSE
To investigate the biological effects of preservative-free artificial tear drops on cultured human corneal epithelial cells in vitro.
METHODS
The effects of the preservative-free artificial tear drops (Kynex(R) 0.1%, Kynex II(R) 0.18% [Alcon, Seoul, Korea] and Hyaluni eye drops(R) 0.15%, 0.3% [Taejun, Seoul, Korea]) on the human corneal epithelial cells were evaluated. An methyl thiazolyl tetrazolium (MTT)-based colorimetric assay was performed to assess the cellular metabolic activity and a lactate dehydrogenase (LDH) leakage assay was used to determine cellular toxicity. The eye drop ingredients were analyzed for electrolyte composition, pH, and osmolarity. We performed a scratch assay and cellular morphology test using electronic microscopy.
RESULTS
The metabolic activity of corneal epithelial cells was higher than controls at 24 hours after exposure and then decreased at 48 and 72 hours after exposure (p < 0.05). The LDH titers of the 4 eye drops were higher compared with controls (p < 0.05). Sodium hyaluronate 0.18% contained lower concentrations of Na+ or Cl- and showed lower osmolarity values compared with the other eye drops. The cellular migration based on the scratch assay was more delayed and cellular damage such as loss of microvilli, rough endothelial reticulum (RER), and mitochondria dilatation was greater than controls based on electron microscopy.
CONCLUSIONS
Long-term exposure to preservative-free sodium hyaluronate eye drops may induce decreased metabolic activity and cellular damage. Thus, preservative-free artificial tears should be used carefully to prevent cellular toxicity.

Keyword

Artificial tear; Cornea; Epithelium; Hyaluronate; Keratocyte

MeSH Terms

Cornea
Dilatation
Epithelial Cells*
Epithelium
Humans*
Hyaluronic Acid*
Hydrogen-Ion Concentration
L-Lactate Dehydrogenase
Microscopy
Microscopy, Electron
Microvilli
Mitochondria
Ophthalmic Solutions
Osmolar Concentration
Reticulum
Seoul
Sodium*
Tears
Hyaluronic Acid
L-Lactate Dehydrogenase
Ophthalmic Solutions
Sodium

Figure

• 

  Figure 1. The absorption rate of the water insoluble formazan dye in corneal epithelial cell exposed in four presevetive-free sodium hyaluronate artificial tear drops-Kynex®, Hyaluni®0.15%, Kynex II®, Hyaluni® 0.3%, and control is Dulbecco Modified Eagle Medium (DMEM)– by scanning spectrometer (ELISA reader). There were significantly differences between control and eye drops at 48 hours and 72 hours after exposure. hr = hours. * p < 0.05.

• 

  Figure 2. Mean LDH titer of cultured corneal epithelial cell exposured four preservative free hyalunate eye drops-Kynex®, Hyaluni eye drops® 0.15%, Kynex II®, Hyaluni eye drops®0.3% and DMEM. There were significantly differences be-tween control and eye drops all the times. LDH = lactate de-hydrogenase; DMEM = Dulbecco Modified Eagle Medium; hr = hours.

• 

  Figure 3. Scratch assay of corneal epithelial cells after exposure to (A) DMEM as control, (B) Kynex®, (C) Hyaluni® 0.15%, (D) Kynex II®, and (E) Hyaluni® 0.3% . Row 1 are result immediately after exposure, Row 2 are after 24 hours, Row 3 are after 48 hours, Row 4 are after 72 hours. The wound closure after exposure of sodium hyaluronate artificial tear were less prominent than control all the time. DMEM = Dulbecco Modified Eagle Medium.

• 

  Figure 4. Transmission electron micrographs of corneal epithelial cells (×8,000). First line were appeared after 24-hours exposure and second lines were after 72-hours exposure to (A) control (DMEM), (B) Kynex®, (C) hyaluni eye drops® 0.15%, (D) Kynex II®,(E) hyaluni eye drops 0.3%. DMEM = Dulbecco Modified Eagle Medium.

Reference

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