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Abstract
Purpose
To evaluate the biological effects and cytotoxicity of gel-type artificial tears on human corneal keratocytes and conjunctival cells in vitro.
Methods
Human corneal keratocytes and conjunctival epithelial cells were exposed to Soothe® and Systane® at variable concentrations. Evaluations were conducted through an MTT-based calorimetric assay to measure the metabolic activity and through a lactate dehydrogenase (LDH) assay to assess cellular damage. Apoptotic response was examined using fluorescent microscopy and flow cytometric analysis, and cellular morphologic results were evaluated with a transmission electron microscope.
Results
The inhibitory effects of corneal keratocyte and conjunctival cell proliferations increased at higher concentrations and longer exposure times to Soothe® and Systane®. The LDH titers increased after Soothe® exposure, but showed no significant difference after Systane® exposure. Soothe® and Systane® treatments both produced fluorescence, representing apoptotic cells. In flow cytometry, the maximal apoptotic response was observed for both types of artificial tears, although Systane® showed less edema, as well as reduced cytoplasmic and nuclear cell degeneration compared to those of Soothe®.
Conclusions
The apoptotic responses of Soothe® and Systane® are associated with inhibitory effects of human corneal keratocyte and conjunctival epithelial cell proliferations. To inhibit the cellular proliferation of human corneal keratocytes and conjunctival epithelial cells, Systane® may be less severe than Soothe® at higher concentrations and longer exposure times.
References
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Figure 1.
The absorption rate of the water-insolubale formazan dye in corneal keratocytes exposed in Soothe® and Systane® by a scanning spectrometer (ELISA reader). Metabolic activity of keratocytes was decreased, at the higher concentration and longer the exposure duration in both artificial tears. The survival rate of Systane® was higher value than that of Soothe®.
Figure 2.
Metabolic activities of cultured human conjunctival epithelial cells showed the dose-response and time-response. Soothe® has more inhibitory effect than Systane®.
Figure 3.
LDH activities of cultured human corneal keratocytes showed that Systane® didn't affect LDH activity significantly, but Soothe® has showed that by dose and time-de-pendent response relationship.
Figure 4.
LDH activities of cultured human conjunctival epithelial cells in Soothe® and Systane®. Soothe® showed greater LDH titer of the conjunctival cells than that of Systane®, especially in the rate of dilution of 20 and 30% after 12 hours exposure.
Figure 5.
Flow cytometric analysis of apoptotic cells using Annexin V-FITC at 30 minutes after exposure. Human corneal keratocytes were left untreated (A) or were treated with 10% (B), 20% (C), and 30% (D) by Soothe®, were treated with 10% (E), 20% (F), and 30% (G) by Systane®. After 30 minutes exposure, most corneal keratocytes in both artificial tears were primarily Annexin V and PI negative.
Figure 6.
Flow cytometric analysis of apoptotic cells using Annexin V-FITC at 4 hours after exposure. Human corneal keratocytes were left untreated (A) or were treated with 10% (B), 20% (C), and 30% (D) by Soothe®, were treated with 10% (E), 20% (F), and 30% (G) by Systane®. After 20% Soothe®, 30% Soothe® and 30% Systane® exposure, a number of cells are positive Annexin V and negative PI (C, D, G) indicating that the cells were in the stage of apoptosis.
Figure 7.
Fluorescent microscopic findings of human conjunctival epithelial cells (A, B, C) and corneal keratocytes (D, E, F) at 4 hours after exposure (original magnification, ×200). (A, D) control. (B, E) 10% Soothe®. (C, F) 30% Systane®. After treating with Soothe® and Systane®, apoptotic cells were visible, which represent fluorescence after binding DNA flowing into cytoplasm due to disruption of nuclear membrane.
Figure 8.
Transmission electron micrograph of human corneal keratocytes (A, B, C) and human conjunctival epithelial cell (D, E, F) appeared after 4 hours exposure to (A, D) control, (B, E) 20% Soothe®, (C, F) 30% Systane®. (Bar length 2 μ m, original magnification, ×3,000∼4,000). In general, the plasma membrane with microvilli (black arrow), nuclear membrane, and nuclei of corneal keratocytes were visible. 20% Soothe® had more severe and damaged cellular structures, such as the plasma membranes with microvilli being disrupted (white arrow), vacuoles enlarge (black arrow head). The chromatin of the nuclear remnant had condensed along the nuclear periphery (white arrow head). The keratocyte and conjunctival epithelial cell showed loss of microvilli, vacuoles formation suspected swollen cytoplasmic organelles at the cytoplasm (black arrow head) compared with control after 4 hours exposure to 30% Systane®.
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