Synergistic Effect of Substance P with Insulin and Insulin-Like Growth Factor-I on Epithelial Migration of the Transformed Human Corneal Epithelial Cells (SV-40)

Round 1

Reviewer 1 Report

The authors have limited themselves to a brief explanation of SP in the introduction and discussion section, while they could explain the origin of SP and its chemical structure and even why they chose SP, along with IGF-1 and insulin.

The graphs shown in figures 2 and 3 are not of sufficient quality and the data trends are unclear.

The graph shown in Figure 5 has no error bars.

There is no image of the morphology of the studied cells.

The authors could have improved the quality of their work by providing a schematic diagram of how to prepare silicon for the study of cell migration.

Author Response

The authors have limited themselves to a brief explanation of SP in the introduction and discussion section, while they could explain the origin of SP and its chemical structure and even why they chose SP, along with IGF-1 and insulin.

- We appreciate the reviewer’s comments. Previously it had been shown that SP effected the migration of rabbit corneal epithelium (references 27, 28). We have previously shown that IGF-1 and SP effect corneal epithelial regeneration.  We added information on the origin of SP and its chemical structure. We also added information why we chose SP along with IGF-1 and insulin.

The graphs shown in figures 2 and 3 are not of sufficient quality and the data trends are unclear.

We appreciate the reviewer’s comments. We added color to the graph lines and expanded the graphs to improve clarity for both figures 2 and 3.

The graph shown in Figure 5 has no error bars.

We appreciate the reviewer’s comments. We added error bars to figure 5

There is no image of the morphology of the studied cells.

We appreciate the reviewer’s comment. We added a picture of cell morphology to the manuscript.

The authors could have improved the quality of their work by providing a schematic diagram of how to prepare silicon for the study of cell migration.

We appreciate the reviewer’s comment. We added a schematic of the methodology to the manuscript.

Reviewer 2 Report

Material and methods should be much better explained.

I believe this articule needs a great review. Methodolody should be much improved. Its not well explained the performance of the study.  Sample size and staristical power doesnt seem to be calculated.  References are apropiated but very old, and some more reciente can be found.

 

Author Response

Material and methods should be much better explained.

-We appreciate the reviewer’s comments. We added a few changes to the methodology as well as adding a figure to help demonstrate the experimental procedure.

I believe this article needs a great review.

-We appreciate the reviewer’s comment. We added additional information to the introduction to provide context and background for our experiments. We also add additional information to the discussion of the paper to help expand its content.

Methodology should be much improved. It’s not well explained the performance of the study. 

-We appreciate the reviewer’s comments. We added a few changes to the methodology as well as adding a figure to help demonstrate the experimental procedure.

Sample size and statistical power doesn’t seem to be calculated. 

-We appreciate the reviewer’s comments. We added extra details below the figures to demonstrate how the statistical significance was represented. In addition, our methodology mentioned that the experiment was done in triplicates and was analyzed using a student T-test.

References are appropriate but very old, and some more recent can be found.

-We appreciate the reviewer’s comment. We added additional information to the manuscript that included more recent citations. The older citations were included to refer to the original studies and show the context of our experiments.

Reviewer 3 Report

Methods: Key details to understand the methodology are missing?  Please provide additional details on how measurements were done.  What measurements of migration were made?  Was the migration distance for individual cells (possible if you measured frequently), or for migration across the dish halfway point (if you just measured at the day timepoints shown in Fig3 for instance)?  Was it the total distance actually a measure of?  In addition to the statistical commentary, please provide a detailed description which would be used to repeat your analysis with the images you took in the experiment.

Please describe generally why this system should be considered representative of a the movement of corneal epithelial cells generally.  How might it be different from the living system context?  Basically, can you provide references or argument for the validity of the system/approach?

Fig2. Please note units of migration distance.  Also note in both the text and figure caption what the time point used to assess this was.

Fig3. Please note units of migration distance.  Also, please reconsider the design of this plot.  As done, the error bars are impossible to read and at best provide only a general sense of the error for a couple of the curves.  Consider using colors, separating plots, etc. to improve clarity.

Fig4. Same comments as Fig3 regarding labeling the y-axis and the plotting of the error bars.

Fig5-8. Same comment about units on the y-axis.

 

FigS8 & FigS9. Error bars are partially occulded by the bar plots

Author Response

details on how measurements were done. What measurements of migration were made? Was the migration distance for individual cells (possible if you measured frequently), or for migration across the dish halfway point (if you just measured at the day timepoints shown in Fig3 for instance)? Was it the total distance actually a measure of? In addition to the statistical commentary, please provide a detailed description which would be used to repeat your analysis with the images you took in the experiment. -We appreciate the reviewer’s comment. Here are our responses to the reviewer’s comments. 1. The measurements were made in mm 2. The migration distance was made in three equal portions from the starting point of the migration to the edge. The mean and standard deviation of three distances were recorded. The distance was to give an average of the entire migration, not individual cells. The total distance was not measured. 3. To repeat the analysis, we would use a microscopic camera to take pictures of the cell migration at the same time and place each day the recording was taken for reference. Please describe generally why this system should be considered representative of a the movement of corneal epithelial cells generally. How might it be different from the living system context? Basically, can you provide references or argument for the validity of the system/approach? -We appreciate the reviewer’s comment. This system was chosen due to previous studies that have been published, which we referenced from Nishida et al. Nishida’s studies utilized in vivo models. We wanted to use something simpler to look at the general trends of SP and its inhibitor on human primary cells. The studies by Nishida et al were done using rabbit models. We also published a review that showed similar efficacy with substance for corneal wound healing in both dogs and humans. Therefore, we wanted to explore this effect mechanistically with human corneal cells (Kopel, J.; Keshvani, C.; Mitchell, K.; Reid, T. The Activity of Substance P (SP) on the Corneal Epithelium. J. Clin. Transl. Ophthalmol. 2023, 1, 35-51. https://doi.org/10.3390/jcto1020006). The references are shown below. Ofuji, K.; Nakamura, M.; Nishida, T. Signaling regulation for synergistic effects of substance P and insulin-like growth factor-1 or epidermal growth factor on corneal epithelial migration. Jpn J Ophthalmol 2000, 44, 1-8, doi:10.1016/s0021-5155(99)00168-9. Nakamura, M.; Ofuji, K.; Chikama, T.; Nishida, T. Combined effects of substance P and insulin-like growth factor-1 on corneal epithelial wound closure of rabbit in vivo. Curr Eye Res 1997, 16, 275-278, doi:10.1076/ceyr.16.3.275.15409. Nakamura, M.; Ofuji, K.; Chikama, T.; Nishida, T. The NK1 receptor and its participation in the synergistic enhancement of corneal epithelial migration by substance P and insulin-like growth factor-1. Br J Pharmacol 1997, 120, 547-552, doi:10.1038/sj.bjp.0700923. Nakamura, M.; Chikama, T.; Nishida, T. Synergistic effect with Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1 on epithelial wound healing of rabbit cornea. Br J Pharmacol 1999, 127, 489-497, doi:10.1038/sj.bjp.0702550. Yamada, N.; Yanai, R.; Kawamoto, K.; Nagano, T.; Nakamura, M.; Inui, M.; Nishida, T. Promotion of corneal epithelial wound healing by a tetrapeptide (SSSR) derived from IGF-1. Invest Ophthalmol Vis Sci 2006, 47, 3286-3292, doi:10.1167/iovs.05-1205. Yamada, N.; Yanai, R.; Nakamura, M.; Inui, M.; Nishida, T. Role of the C domain of IGFs in synergistic promotion, with a substance P-derived peptide, of rabbit corneal epithelial wound healing. Invest Ophthalmol Vis Sci 2004, 45, 1125-1131, doi:10.1167/iovs.03-0626. Nakamura, M.; Nishida, T.; Ofuji, K.; Reid, T.W.; Mannis, M.J.; Murphy, C.J. Synergistic effect of substance P with epidermal growth factor on epithelial migration in rabbit cornea. Exp Eye Res 1997, 65, 321-329, doi:10.1006/exer.1997.0345. Nishida, T.; Nakamura, M.; Konma, T.; Ofuji, K.; Nagano, K.; Tanaka, T.; Enoki, M.; Reid, T.W.; Brown, S.M.; Murphy, C.J.; et al. [Neurotrophic keratopathy--studies on substance P and the clinical significance of corneal sensation]. Nippon Ganka Gakkai Zasshi 1997, 101, 948-974. Fig2. Please note units of migration distance. Also note in both the text and figure caption what the time point used to assess this was. -We appreciate the reviewer’s comment. We mentioned that the measurements were done on day 7. Fig3. Please note units of migration distance. Also, please reconsider the design of this plot. As done, the error bars are impossible to read and at best provide only a general sense of the error for a couple of the curves. Consider using colors, separating plots, etc. to improve clarity. -We appreciate the reviewer’s comments. We removed some of the trend lines to improve clarity. Fig4. Same comments as Fig3 regarding labeling the y-axis and the plotting of the error bars. -We appreciate the reviewer’s comments. We removed some of the trend lines to improve clarity. Fig5-8. Same comment about units on the y-axis. -We appreciate the reviewer’s comments. We added units to the y-axis FigS8 & FigS9. Error bars are partially occulded by the bar plots We appreciate the review’s comment. We made sure the error bars were not occluded. I believe the figures that showed this was Fig 7 and 8.

Round 2

Reviewer 1 Report

The authors have addressed the comments and the quality of the report has improved. The article seems to be sound enough for publication.

Author Response

Thank you

Reviewer 2 Report

As a formerly said the design of the study should be improved. no sample size nor statistical power have been formerly calculated, and no matter the introduction and conclusions have been improved this article needs much more improvement.

 

Author Response

Reviewer 2 As a formerly said the design of the study should be improved. no sample size nor statistical power have been formerly calculated, and no matter the introduction and conclusions have been improved this article needs much more improvement. -We appreciate the reviewer’s comment. The sample size for the study was three determinants as mentioned in the figure diagrams. The power is not required for this study since it is not a clinical trial nor were any in vivo animals or human subjects were used. We also improved the description of the manuscript by adding more details per the other reviewer regarding the method of measurement and how this was done with regards to statistical analysis.