Evaluation of protection against desiccation with different articial tears containing 0.1%-0.3% hyaluronic acid in experimental dry eye model

Background: Tear lm instability, hyperosmolarity, ocular surface inammation, apoptosis and neuro-sensory abnormalities are causes of dry eye. Lubricant target tear lm instability, the most effective agent for tear lm stabilization being Sodium Hyaluronate 0.1 -0.3%. Sodium Hyaluronate is suggested to be protective for epithelium in dry eye. To test this hypothesis, this study was performed in vitro with commercially available solutions containing hyaluronic acid (HA) in concentrations ranging from 0.1 to 0.3%. To evaluate the desiccation protection capability of different Sodium Hyaluronate 0.1 to 0.3%, we employed a reproducible in vitro cell culture system.

solutions. Greatest protection was observed at 15 minutes with most agents. Best results in protection from desiccation was assessed with sample 2 even at maximum exposure time at 45 minutes. Sample 2 showed an average survival rate of 91% at 45 minutes exposure time, whereas no signi cant amount of vital cells were detected after application with sample 6. Sample 6 was the only substance that presented with early signi cant cell loss at 0 and 15 minutes by 35%.
Conclusions: Higher concentration of Hyaluronate acid with 0.3% and an ionic composition close to the normal tear uid seem to provide the best protective effect against desiccation in experimental dry eye.

Background:
Dry eye syndrome, predominantly affecting post-menopausal women, is a disorder of the ocular surface, resulting in irritation, pain, burning sensation, discomfort and decreased visual acuity and consecutively leading to depression and anxiety disorders. Tear lm instability, hyperosmolarity, ocular surface in ammation, apoptosis and neuro-sensory abnormalities play an important role in the development of dry eye disease. One of the most effective agent for moistening of the ocular surface is the application of arti cial tears with hyaluronic acid. Several experimental and clinical studies revealed the bene cial use of hyaluronic acid based arti cial tears in terms of symptom control as well as causal treatment (2-4, 7, 9, 13). This study`s aim was to evaluate the protective effect of ve commercial unpreserved ophthalmic solutions, containing various concentrations of hyaluronic acid and differing compositions in order to improve the current therapeutic options of dry eye syndrome. For this purpose, an in-vitro model of humanized epithelial conjunctival and corneal cells was used, to analyze the effects of the eye drops against desiccation.

Methods:
Cell cultures: Two different epithelial cell cultures were used to analyze the protection of the arti cial tears against desiccation. The conjunctival cell line Chang 1-5c-4 (American Type Culture Collection) was Chemie GmbH) and 1% penicillin/streptomycin (GIBCO®/Thermo Fisher Scienti c). At least two hours before incubation at 37 °C and 5% CO 2 , the asks were precoated with a mixture containing 0.01 mg/mL bronectin, 0.03 mg/mL bovine collagen type I (both Sigma-Aldrich Chemie GmbH) and 0.01 mg/mL bovine serum albumin (GIBCO®/Thermo Fisher Scienti c). Viability testing The con uent growing cells were incubated in 96-well-plates with 50 µL of each sample (see Table 1) was applied on six deepenings. The same amount of PBS served as negative control, while 100 µL of unsupplemented medium was the positive control. After 20 minutes, the medications were cautiously removed and the cells were exposed to a continuous air ow for 0, 15, 30 and 45 minutes. To assess the amount of viable cells, the cells were again incubated for four hours at 37 °C with a mixture containing 100 µL respective medium and 10 µL alamarBlue®. The absorption of the oxidized form of alamarBlue® (ThermoFischer Scienti c) was measured at wave length 570 nm and 600 nm using an ELISA-reader.
This procedure was repeated four times, so that 24 measured values were available for each sample. To validate the results, another test, the LIVE/DEAD® Viability/ Cytotoxicity Kit was performed. After exposure to continuous air ow, 100 µL PBS and 100 µL PBS based mixed solution composed of 2 µl/mL 1,5 mM propidium iodide and 0.5 µl/mL 4 mM Calcein AM were applied on the cells. After 20 minutes the greenly stained viable cells and redly stained dead cells could easily be identi ed and counted under the uorescence microscope (Olympus IX, Scion VisiCapture).

Analysis:
The survival rate for the alamarBlue® assay was assessed using the following formula:  It contains the highest to tested concentration (0.3%) and most closely resembles normal human tear lm in terms of osmolarity, pH and ratio of electrolytes (2, 6, 11).
Even after increasing drying time up 45 minutes, no further cell loss was observed. This implies that the higher the concentration of hyaluronic acid, the better the protective effect against desiccation.
Incubation with sample 6 lead to loss of viable cells at 0 minutes in both alamarBlue® and LIVE/DEAD® Viability/ Cytotoxicity Kit. This could be due to lack of protective properties, but more likely due to immediate cytotoxic effect during the twenty minute application time.
Generally speaking, samples 1-5 had similar survival rates throughout this study. This is due to similar pharmacological properties. The molecular weight and osmolality of each product lead to the shown differences.
Generally speaking, samples 1-5 had similar survival rates throughout this study. This is due to similar pharmacological properties. The molecular weight, osmolality and viscosity of each lead to the shown differences. The composition of sample 6 was unknown, but it was apparent that it had a higher viscosity. The di culties in removing the solution after application and the consequential higher mechanical cell loss could also be an explanation for the lower survival rates. All samples except sample 6 had highest protective effects at 15 minutes of stress exposure.
The cell model was performed previously and was found to be suitable to assess cytotoxic effects of wetting agents. Various preserved and unpreserved products were tested in studies, but none of them containing hyaluronic acid. The tested agents had lower survival rates compared to the samples in this study.
Previous studies showed that hyaluronic acid has a positive effect on dry eyes. (4,9). It was also observed that combinations with trehalose (10) or vitamin B12 (5) were more protective then simple hyaluronic acid and lead to reduction of in ammatory processes due to synergistic effects.
A conjunctival and corneal cell line was used in this study (1,8). The different cell reaction lead to conclude that in vivo the different parts of the ocular surface will also present with different reactions. Cell cultures cannot predict exactly the reactions in vivo due to lack of consideration of blood supply, drug effusion and mucin production. Furthermore it is impossible to consider the physiological interactions on the ocular surface in vitro, hence further clinical evaluation is needed here.

Conclusion:
The results suggest that high concentrations of hyaluronic acid (sample 2 hyaluronic acid 0.3%) and an ionic composition to the normal tear uid in arti cial tears provide the best protective effect against in vitro desiccation. Additive clinical trials are needed for further validation. Availibilty of data and materials Datasets used and analysed during the current study are available from the corresponding author on reasonable request. All data generated and analysed during this study are included in this published article and its supplementary information les. Figure 1 The   This gure shows vital (greenish) and avital (reddisch) cells of the conjunctival cell line after application of sample 2 after 30 minutes exposure. The cell count of viable cells signi cantly outweighs the number of avital cells.