Blue Light-Based Method to Induce Oxidative Stress on Rabbit Corneal Epithelial (RCE) Cells: Development and Validation
, Mariacristina Di Gangi 1, Patrizia Chetoni 1,2, Silvia Tampucci 1,2,3,*, Daniela Monti 1,2,3,‡ and Susi Burgalassi 1,2,‡Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors cover a very important and interesting topic in the article. However, it requires several additions.
What is the survival of RCE cells under the influence of antioxidants and irradiation simultaneously?
In Figures 2 and 3, please add the level of control cells not treated with irradiation. Next, please state whether the changes in individual doses and times are statistically significant compared to the control samples.
Author Response
We sincerely thank Reviewer #1 for the thorough evaluation of our manuscript and for the valuable comments provided. The suggestions have been carefully addressed, and the manuscript has been revised accordingly. We believe that these modifications have strengthened the clarity and overall quality of the work.
Please find below our detailed responses to each comment.
Comment 1. What is the survival of RCE cells under the influence of antioxidants and irradiation simultaneously?
Response 1. The irradiation conditions selected in this study (4.57 W/m² for 30 minutes) were previously demonstrated to be non-cytotoxic, as confirmed by WST-1 assays performed immediately and after a 24-hour recovery period. The model was intentionally established under sublethal conditions in order to investigate oxidative stress responses without inducing overt cell death. In the combined condition (antioxidant treatment during irradiation), intracellular ROS levels were significantly reduced compared with irradiated controls. Since irradiation alone did not affect cell viability and antioxidant treatment attenuated oxidative stress rather than enhancing it, no reduction in cell survival is expected under simultaneous exposure. The primary endpoint of this study was the modulation of oxidative stress under controlled sublethal irradiation, and the data consistently indicate that the tested antioxidants act at the redox level without evidence of cytotoxic effects under the applied conditions.
Comment 2. In Figures 2 and 3, please add the level of control cells not treated with irradiation.
Response 2. In Figures 2 and 3, cell viability values are expressed as a percentage relative to the non-irradiated control, which was set to 100% by definition. Therefore, the control condition is implicitly represented in the normalization procedure and would appear as a constant 100% reference value in the graphs. To avoid redundancy in data presentation, the control bar was not displayed separately but we have specified it in the revised manuscript.
Comment 3. Next, please state whether the changes in individual doses and times are statistically significant compared to the control samples.
Response 3. The text has been modified according to the reviewer's comment (section 3.2)
Reviewer 2 Report
Comments and Suggestions for AuthorsThe article addresses a relevant problem of the development and validation of a reproducible model of blue light-induced oxidative stress in rabbit corneal epithelial cells . The research topic is timely in the context of the growing use of digital devices and their potential impact on the ocular surface. However, there are a number of minor comments:
1) What is the rationale for choosing a wavelength of 405 nm? Why was 450 nm not selected? The 405 nm wavelength lies at the boundary of the blue and violet ranges and exhibits significantly higher photochemical activity. The choice of this wavelength should be justified in relation to the actual emission spectrum of digital displays.
2) In section 2.2.4, it is stated that the ROS assay with DCF-DA was performed 24 hours after irradiation. Why was it conducted at 24 hours rather than immediately? The signal may have dissipated by that time.
3)What was the number of replicates used in the experiments?
4)Cells were irradiated at RT outside the CO2 incubator. How do the authors ensure that the pH of the medium, which depends on the CO2/bicarbonate buffer system ,did not change over the 30–180 minute exposure period, and how might this have affected the cellular response?
5)Blu Yal A Free and Thealoz were diluted 1:1 with growth medium. What is the rationale for using this dilution ratio?
6) On what basis were the concentrations of AA (0.1 mg/mL) and OLE (0.2 mg/mL) selected?
7)In Figure 8, ROS levels are shown for PRE/POST treatment with commercial products; however, a separate "irradiation without any additives" control within the same experimental series is absent for direct comparison.
Overall, the article represents a practically significant work, making a real contribution to the development of in vitro models for screening antioxidant compounds and ophthalmic formulations. After addressing these minor issues, the manuscript may be recommended for publication in Scientia Pharmaceutica.
Author Response
We thank Reviewer #2 for the valuable comments and constructive feedback. The manuscript has been revised in response to all observations, and we believe that these modifications have strengthened the work.
Please find below our detailed responses to each comment.
Comment 1. What is the rationale for choosing a wavelength of 405 nm? Why was 450 nm not selected? The 405 nm wavelength lies at the boundary of the blue and violet ranges and exhibits significantly higher photochemical activity. The choice of this wavelength should be justified in relation to the actual emission spectrum of digital displays.
Response 1. The text has been modified according to the reviewer's comment (section 2.2.1)
Comment 2. In section 2.2.4, it is stated that the ROS assay with DCF-DA was performed 24 hours after irradiation. Why was it conducted at 24 hours rather than immediately? The signal may have dissipated by that time.
Response 2. The text has been modified according to the reviewer's comment (section 3.3)
Comment 3. What was the number of replicates used in the experiments?
Response 3. “All experiments were performed in at least six independent biological replicates” was added in section 2.2.5
Comment 4. Cells were irradiated at RT outside the CO2 incubator. How do the authors ensure that the pH of the medium, which depends on the CO2/bicarbonate buffer system, did not change over the 30–180 minute exposure period, and how might this have affected the cellular response?
Response 4. Both irradiated and non-irradiated control samples were maintained outside the COâ‚‚ incubator for the same exposure times, ensuring identical environmental conditions across all groups. Although bicarbonate-buffered culture media are optimized for 5% COâ‚‚, the relatively short exposure periods (30–180 minutes) are unlikely to induce biologically relevant pH alterations. Importantly, no significant differences in cell viability were observed between irradiated and non-irradiated samples at any time point. Furthermore, the optical density (OD) values of untreated controls were consistent with those routinely obtained under standard incubator conditions in our laboratory. Then, we can assume that temporary removal from the COâ‚‚-controlled atmosphere did not introduce confounding effects affecting cellular viability.
Comment 5. Blu Yal A Free and Thealoz were diluted 1:1 with growth medium. What is the rationale for using this dilution ratio?
Response 5. The 1:1 dilution represented the highest feasible product concentration that allowed maintenance of cell viability and physiological culture conditions. A comment was included at the end of section 2.2.4.
Comment 6. On what basis were the concentrations of AA (0.1 mg/mL) and OLE (0.2 mg/mL) selected?
Response 6. The concentrations of ascorbic acid and oleuropein (were selected based on previously published literature. A comment was included in section 2.2.4.
Comment 7. In Figure 8, ROS levels are shown for PRE/POST treatment with commercial products; however, a separate "irradiation without any additives" control within the same experimental series is absent for direct comparison.
Response 7. An irradiated control without additives (light control) was included in the same experimental series and has now been added to Figure 8 to allow direct comparison within the same plate. Moreover, a comment was added in the relevant section.
