Coenzyme Q10 and Fish Oil Supplementation for Reducing Retinal Oxidative Stress in a Rat Model
Round 1
Reviewer 1 Report
The authors have already published a paper, always using the same animal model, where they demonstrate that CoQ10 supplementation decreased superoxide dismutase (SOD) and glutathione, but increased catalase, suggesting reduced oxidative stress (Beharry KD, Cai CL, Henry MM, Chowdhury S, Valencia GB, Aranda JV. Co-Enzyme Q10 and n-3 Polyunsaturated Fatty Acid Supplementation Reverse Intermittent Hypoxia-Induced Growth Restriction and Improved Antioxidant Profiles in Neonatal Rats. Antioxidants (Basel). 2017 Dec 16; 6 (4): 103 . doi: 10.3390 / antiox6040103) and a work where supplementation with CoQ10 improves the characteristics of OIR (Beharry KD, Cai CL, Siddiqui F, Chowdhury S, D'Agrosa C, Valencia GB, Aranda JV. Comparative Effects of Coenzyme Q10 or n-3 Polyunsaturated Fatty Acid Supplementation on Retinal Angiogenesis in a Rat Model of Oxygen-Induced Retinopathy. Antioxidants (Basel). 2018 Nov 9; 7 (11): 160. doi: 10.3390 / antiox7110160).
In this manuscript, the authors compare the antioxidant efficacy of CoQ10 and fish oil in two models of intermittent hypoxia (50/12 and 21/12) and demonstrate that supplementation with fish oil improved growth, but CoQ10 provided superior benefits for reducing IH-induced oxidative stress and retinopathy.
Authors should verify, by electroretinogram, whether these treatments result in a better functional response.
In figure 1 the authors show sections of the retina relative to the various groups. In particular, they show that the retinal structure in the two models of HA is altered, especially in the 21/12 group, where they observe a great distortion of the INL with the presence of hemorrhages (panel G). Is it bleeding or abnormal neovessel groth? Furthermore, in the legend the authors claim to observe pathological changes also in the ONL (in the figure it is not observed).
The retinal sections in figure 7 appear to be taken in different retinal areas.
Figures 1 and 7 are missing the calibration bars
Author Response
- Authors should verify, by electroretinogram, whether these treatments result in a better functional response.
Response: We thank the reviewer for this recommendation. We agree that an electroretinogram would provide key information regarding retinal function. Unfortunately, we do not have access to such equipment at our institution. It is not feasible for us to send the rats out to another laboratory at another institution, to assess the retinal function because:1) assessment should be done immediately post IH exposure; and 2) there are strict State and Institutional regulations regarding transport of animals from facility to facility. Since our animals were housed for several weeks at our facility, the other facility would not accept them due to possible contamination.
- In Figure 1 the authors show sections of the retina relative to the various groups. In particular, they show that the retinal structure in the two models of IH is altered, especially in the 21/12 group, where they observe a great distortion of the INL with the presence of hemorrhages (panel G). Is it bleeding or abnormal neovessel growth? Furthermore, in the legend the authors claim to observe pathological changes also in the ONL (in the figure it is not observed).
Response: The author is correct regarding the severe abnormalities of the neural retina, particularly in the group exposed to 21/12% O2. This could be due to hemorrhage, however, early studies by Ashton N, et al. (Br. J Ophthalmol, 1954) showed a similar phenomenon in the retina of kittens exposed to hyperoxia. The author stated that it was diffuse proliferation of mesenchymal cells in the inner layers of the retina. The distorted and folded retina was indicative of retinal detachment. Animal studies by Kim B et al. Sci Rep 2017 showed similar malformed ONL which was attributed to severe photoreceptor death and retinal gliosis, also indicative of retinal detachment in mice. Studies in rats administered an intravitreal VEGF blocker also showed distorted INL and ONL suggesting severe apoptotic damage (Romano MR et al. Brain Res. 2012). These previous reports demonstrate that distortions in the INL and ONL may indicate severe photoreceptor damage and retinal detachment. This is further discussed in the revised manuscript and we have added these references to support our statements (references 34-36).
The reviewer is correct regarding damage to the INL, not the ONL. This was a typographical error. We have corrected the text in the legend.
- The retinal sections in figure 7 appear to be taken in different retinal areas.
Response: We have corrected the images for consistency. We mainly focused on the area closest to the optic nerve for all images.
- Figures 1 and 7 are missing the calibration bars.
Response: The scale bars of the figures were placed at the side or top of the image when we oriented the images for consistency or removed due to cropping for presentation. We have now added the scale bars at the bottom of all the figures.
Reviewer 2 Report
The manuscript demonstrated that exposure to intermittent hypoxia (IH) causes severe retinopathy regardless of recovery/reoxygenation in hyperoxia or room air. The authors also studied the effect of IH with and without fish oil and CoQ10 on oxidative stress, apoptosis, and angiogenesis in retina. Early supplementation with fish oil or CoQ10 was beneficial, but that CoQ10 showed superior results regarding reducing IH-induced retinal oxidative stress and injury.
My comments
-In abstract: "Supplementation with fish oil improved growth": which growth? please verify
-Introduction needs to be improved, especially the first part: " The prevalence of blindness from ROP is about 50,000" is it worldwide? Also, the role of low birth weight and prematurity were mentioned many times
-In materials and methods: all catalogue numbers for all kits and reagents used should be indicated
-For immunofluorescence results: add scale bar for figures
- Why were not gene analyses and immunofluorescence results confirmed by western blot? Also, intensity of immunofluorescence can be analysed.
Author Response
- In abstract: "Supplementation with fish oil improved growth": which growth? please verify.
Response: We referred to overall somatic growth from our previous publication. However, we have corrected the statement regarding growth as data were not presented in this submission. We revised the statement to focus on the subject of this manuscript
- Introduction needs to be improved, especially the first part: " The prevalence of blindness from ROP is about 50,000" is it worldwide? Also, the role of low birth weight and prematurity were mentioned many times.
Response: We thank the reviewer for this recommendation and we have revised the introduction as suggested.
- In materials and methods: all catalogue numbers for all kits and reagents used should be indicated.
Response: All catalogue numbers for the kits and reagents used were provided in the “Method” section.
- For immunofluorescence results: add scale bar for figures
Response: The scale bars of the figures were placed at the side or top of the image when we oriented the images for consistency or removed due to cropping for presentation. We have now added the scale bars at the bottom of all the figures.
Why were not gene analyses and immunofluorescence results confirmed by western blot?
Response: The reviewer raises a good point regarding validation of the gene analyses and IHC results with Western blots. This was definitely considered. However, due to small sample volume, the retinal tissues were already pooled for the ELISA assays. There was no remaining tissue to conduct Western blot assays. The method for extracting protein for Western blots requires the use of RIPA buffer. This was not used for extracting protein for ELISA assays.
- Also, intensity of immunofluorescence can be analyzed.
Response: Quantitation of the immunofluorescence intensity was presented in Table 1.
Reviewer 3 Report
The manuscript by Siddiqui et al investigated efficacy of olive oil and fish oil supplementation in lowering the oxidative stress in the retina. This research area has of great impact now a days and authors addressed the research question very well. Here are some points observed in this manuscript.
1. Article is nicely written and good background literature
2. Statistical analysis done well
3. The authors evaluated an array of oxidative and antioxidative markers to confirm the observations
4. Why ‘Ret-Inal’ in the main title of the manuscript, it seems confusing
5. The histology images in Figure 1 doesn’t look like properly organized and the size aspect ratio of the images are not correct.
6. Figure 1 and Figure 7 captions indicated the details of scale bars but couldn’t find that in the images
the effect of bisretinoid mediated stress on the RPE cells with and without the role of dark and suggests the role in age related macular degeneration. Oxidant stress mediated retinal damage is considered as a major factor in AMD and the study put forth some novel strategies in this regard. Here are some merits and comments on this manuscript.
Author Response
- Article is nicely written and good background literature.
- Statistical analysis done well
- The authors evaluated an array of oxidative and antioxidative markers to confirm the observations
Response: We thank the reviewer for his/her kind comments.
- Why ‘Ret-Inal’ in the main title of the manuscript, it seems confusing.
Response: We thank the reviewer for pointing out this typographical error. This must have happened during the configuration with the Journal template. We have corrected it.
- The histology images in Figure 1 doesn’t look like properly organized and the size aspect ratio of the images are not correct.
Response: In the figures, some images appear smaller (Figures B and E) or larger (Figures D and G) in size due to the effect of oxygen exposure and/or supplementation. All images were captured at the same magnification (40X). In terms of organization, we tried to position the images with the same orientation for consistency. If the images are edematous and swollen (as in Figures D and G), it is difficult to get all the layers in one image without changing the magnification. Treatment with CoQ10 in RA (Figure B) and 50/12% O2 (Figure E) decreased the size of the retinal layers, while supplementation with olive oil increased the sizes in both IH paradigms (Figures D and G).
- Figure 1 and Figure 7 captions indicated the details of scale bars but couldn’t find that in the images
Response: The scale bars of the figures were placed at the side or top of the image when we oriented the images for consistency or removed due to cropping for presentation. We have now added the scale bars at the bottom of all the figures.
Round 2
Reviewer 2 Report
The authors addressed my comments and the manuscript has been improved
Author Response
We thank the reviewer for their acceptance of the changes.
