Beneficial Effect of Sirolimus-Pretreated Mesenchymal Stem Cell Implantation on Diabetic Retinopathy in Rats

We would like to express our sincere gratitude for your invaluable feedback and constructive comments during the revision process of our manuscript. Your insights and suggestions have significantly contributed to the improvement and overall quality of the paper. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1: Authors should explain why they didn't use OCT for DR control.

Response 1: Thank you for pointing this out. We had considered using Optical coherence tomography (OCT); however, in this study, substance administration was carried out after induction of diabetic retinopathy (DR) in rats. Streptozotocin-induced DR typically occurs after prolonged exposure to high blood glucose levels, generally exceeding 150 mg/dL [1]. In this study, DR was confirmed to be induced at the 10-week of diabetes induction. However, according to Kyselova (2005), cataract progression becomes evident in streptozotocin-induced diabetic rats at the 10-week of diabetes induction [2]. Consistent with this research, cataracts were observed in the majority of diabetic rats in our study. The presence of cataracts impedes accurate imaging during OCT as the light dose not effectively penetrate the eye. Due to the same reasons, evaluating retinal angiography was also challenging. Consequently, in this study, OCT was not utilized for retinal assessment; instead, retinal tissues were collected and evaluated histopathologically.

1. Quiroz, J.; Yazdanyar, A. Animal Models of Diabetic Retinopathy. Ann. Transl. Med. 2017, 93, doi:10.21037/atm-20-6737.2. Kyselová, Z.; Garcia, S.; Gajdošíková, A.; Gajdošík, A.; Štefek, M. Temporal Relationship between Lens Protein Oxidation and Cataract Development in Streptozotocin-Induced Diabetic Rats. Physiol. Res. 2005, 54, 49–56, doi:10.33549/physiolres.930613.

Comments 2: Please add arrows or areas on Figure 4(A) to show the locations of interest.

Response 2: Thank you for enhancing the clarity of my figure. We have made modifications based on your comments to emphasize the relevant locations. The Inner Nuclear Layer (INL) has been indicated with a yellow arrow, while the Photoreceptor Layer (PRL) is represented by a white arrow. Additionally, we have upgraded the figure to a higher definition.

We would like to express our sincere gratitude for your invaluable feedback and constructive comments during the revision process of our manuscript. Your insights and suggestions have significantly contributed to the improvement and overall quality of the paper. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

 Comments 1: The authors need to expand on the pathophysiology of Diabetic Retinopathy in the introduction.

Response 1: We appreciate the reviewer’s comment on the lack of pathophysiology of diabetic retinopathy in the introduction. In accordance with the reviewer’s comment, we have revised the manuscript. In the original version, only the microvascular damage caused by hyperglycemia in diabetes, resulting in retinal hypoxia and increased vascular endothelial growth factor (VEGF) levels, was mentioned. In the revised manuscript, various biochemical and metabolic abnormalities associated with diabetic conditions are detailed, leading to retinal hypoxia. As a consequence, an increase in VEGF levels is described, which leads to angiogenesis, increased retinal vessel permeability, and disruption of the barrier between the retina and blood. This modification can be found in the second paragraph on page 2, lines 47-55 of the revised manuscript.

Here's the updated text in the manuscript.

[Exposure to DM conditions leads to various biochemical and metabolic abnormalities, including changes in the redox state of pyridine nucleotides, accumulation of sorbitol, over-activation of protein kinase C, oxidative stress due to excessive free radical production, and alterations in hemodynamics. These pathogenic mechanisms play a crucial role in the progression of DR. Additionally, within weeks of the onset of diabetes, leukostasis occurs in retinal capillary, leading to capillary occlusion and local ischemia [1]. Retinal hypoxia results in increased expression of vascular endothelial growth factor (VEGF) [2]. Elevated levels of VEGF induce angiogenesis and enhance retinal vessel permeability, causing disruption of the barrier between the retina and blood [2,3].

1. Curtis, T.M.; Gardiner, T.A.; Stitt, A.W. Microvascular Lesions of Diabetic Retinopathy: Clues towards Understanding Pathogenesis? Eye 2009, 23, 1496–1508, doi:10.1038/eye.2009.108.
2. Pande, G.S.; Tidake, P. Laser Treatment Modalities for Diabetic Retinopathy. Cureus 2022, 14, e30024, doi:10.7759/cureus.30024.
3. Kollias, A.N.; Ulbig, M.W. Diabetic Retinopathy. Dtsch. Ärzteblatt Int. 2010, 107, 75–83, doi:10.3238/arztebl.2010.0075.

Comments 2:  The authors created a beautiful figure, but the figure in the manuscript is not clear enough. The original or high-definition figures should be provided.

Response 2: We apologize for the poor quality of the figures. We have attached the high-definition figures below and sent the original files via e-mail to [email protected]. Also, I have changed figures in the manuscript to high-definition and add arrows for clarity. This modification can be found in the page 8.

Comments 3:  Scientific articles with several technical terms or acronyms should include an abbreviation list. Abbreviations can improve article reading and comprehension

Response 3: We appreciate the suggestion to include an abbreviation list. We believe that this addition improves the readability and comprehension of our manuscript. Therefore, we have added the abbreviation list in the supplementary material. This can be found in the page 12 of revised manuscript. Additionally, we have attached the list of abbreviations below.

List of abbreviations

Comments 4:  Figures must be self-explanatory (Fig. 3 and Fig. 4), so abbreviations must be explained as footnotes for readers to view without the need to refer to the main text.

Response 4: Thank you for pointing this out. we have added explanation of abbreviations in the foot notes of Figure 1., Figure 3. and Figure 4. These changes can be found in page 5, 7 and 8. 

 Comments 1:  References 4 and 32- authors should not be written with capital letters

Response 1: We appreciate your feedback and have made the corrections to the authors’ names. These changes are reflected on pages 12 and 13. Additionally, following adjustments based on another reviewer’s comments, reference 32 has been revised to reference 30. We have attached the updated contents below.

4. Rakieten, N.; Rakieten, M.L.; Nadkarni, M.V. Studies on the Diabetogenic Action of Strepto-zotocin (NSC-37917). Cancer Chemother. Rep. 1963, 29, 91–98.

30. Vézina, C.; Kudelski, A.; Sehgal, S.N. RAPAMYCIN (AY-22, 989), A NEW ANTIFUNGAL AN-TIBIOTIC. The Journal of Antibiotics 1975, 28, 721–726, doi:10.7164/antibiotics.28.721.