F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.170846.3 Study Protocol Articles Antioxidant Support for the Corneal Endothelium: Evidence from Vitamin C Supplementation in Hard Cataract Phacoemulsification

[version 3; peer review: 2 approved, 1 approved with reservations]

 Widyawati Syska Conceptualization Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-7119-5250 a 1 Sitompul Ratna Resources Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 I Wanandi Septelia Resources Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-7963-8853 2 Louisa Melva Data Curation Formal Analysis Methodology Validation Visualization 3 Kekalih Aria Data Curation Formal Analysis Methodology Supervision 4 LDR Nora Rina Resources Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 Ophthalmology, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10430, Indonesia Biochemistry and Molecular Biology, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10430, Indonesia Pharmacology and Therapeutics, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10430, Indonesia Community Medicine, Faculty of Medicine, University of Indonesia, Jakarta, Jakarta, 10430, Indonesia syska.widyawati@gmail.com

No competing interests were disclosed.

 24 4 2026 2025 14 1117 24 3 2026 Copyright: © 2026 Widyawati S et al. 2026 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Phacoemulsification is one of the most frequent surgeries in the world. However, prolonged use of phacoemulsification machines produces reactive oxygen species which will damage corneal endothelial cells. Ascorbic acid has an antioxidant capacity to neutralize oxidative stress in the anterior chamber. This study will investigate the protective effect of ascorbic acid on corneal endothelial cells in patients with hard nuclear cataracts.

 Methods

This study is a double-blinded randomized controlled trial. Samples will be divided into three groups, and 500 mg vitamin C three times daily (1500 mg/day), or placebo will be received for seven weeks. Clinical characteristics, ascorbic acid, malondialdehyde, and total antioxidant capacity of patients in serum and aqueous humor will be measured before and after intervention and phacoemulsification.

 Conclusions

Data from this study will reveal the protective effect of oral vitamin C supplementation on the corneal endothelial cells in patients with hard nucleus cataracts.

This trial has been registered at ClinicalTrials.gov (identifier: NCT06781970; registered on 17 January 2025). The trial record is available at: https://clinicaltrials.gov/ct2/show/NCT06781970

ascorbic acid phacoemulsification oxidative stress hard nucleus cataract. The author(s) declared that no grants were involved in supporting this work. Amendments from Version 2

The manuscript has been substantially revised to address reviewer comments and strengthen methodological clarity. The Introduction has been refined to better define the knowledge gap by distinguishing current evidence on antioxidant strategies and highlighting the lack of clinical data on systemic (oral) vitamin C supplementation in hard nuclear cataracts, particularly regarding endothelial protection. The rationale for selecting oral vitamin C has been expanded, including comparison with other antioxidant strategies, and justification of the 1500 mg/day dose based on pharmacokinetic evidence demonstrating steady-state plasma levels. A clear study hypothesis has been added. The Methods section now includes a dedicated sample size calculation based on the primary outcome (percentage endothelial cell density loss), specifying the expected difference, standard deviation, alpha, and power. Primary and secondary outcomes have been explicitly defined. The section on randomization, blinding, and allocation concealment has been clarified, including sequence generation, use of sealed opaque envelopes, and masking of participants, surgeons, outcome assessors, and laboratory personnel. Intraoperative parameters (effective phacoemulsification time and duration of surgery) will be recorded and included as covariates. Details regarding aqueous humor sampling (0.2 mL pre- and post-procedure) have been expanded with safety justification and supporting references. The statistical analysis plan has been refined, specifying ANOVA or Kruskal–Wallis tests, post-hoc corrections, and multivariable analysis to control for confounders. Minor revisions include correction of terminology, clarification of dosing (500 mg three times daily), and grammatical improvements. Ethical approval number and date have been added, and a CONSORT flow diagram has been included to illustrate study procedures.

 Introduction

Cataract is the most probable cause of blindness in Indonesia. Universally, WHO estimated that 51% of blindness in the world is caused by cataracts and there are 20 million people who are blind from cataracts. In 2020, the prevalence of blindness in Indonesia is 1.4% with 81.2%, or three million, being from cataracts. This number progressively increases as aging is the risk factor for cataracts and is positively correlated to cataract density. With 10% of the total population in Indonesia being of geriatric population in 2021, it is predicted there will be more than 50 million elders in Indonesia in 2045 suffering from cataracts. 1 , 2

Phacoemulsification is the most used cataract extraction method, with a high success rate of 80.1% compared to extracapsular cataract extraction (ECCE) and intracapsular cataract extraction (ICCE). 3 Phacoemulsification utilizes ultrasonic waves, up to 20,000 Hz, to break apart and emulsify the lens. Mechanic vibration of the probe causes bubbles through the acoustic cavitation phenomenon in the aqueous humor. These bubbles, if popped, will dissociate water molecules and produce reactive free radicals. 3 Phacoemulsification is safer and more effective than other cataract extraction methods. However, higher power and longer duration of ultrasonic waves from the phacoemulsification probe may cause postoperative complications through the increased number of free radicals. 4

Free radicals bind with antioxidants in tissues. The imbalance between the number of free radicals and antioxidants in the tissue causes oxidative stress. 5 On a molecular level, oxidative stress can be measured through various markers, one of which is malondialdehyde (MDA). MDA is the end-product of phospholipid oxidation, in which the phospholipid bond is one of the main targets of reactive oxygen species.

Corneal endothelial cells (CEC) have little to no regeneration capacity, hence CEC damage is irreversible. Naturally, corneal endothelial cells diminish 0.3-0.6% yearly. 6 This phenomenon is aggravated by increased levels of oxidative stress. Phacoemulsification causes a 40% increase in intraocular free radicals if not followed by sufficient antioxidants. Patients with hard nucleus cataracts undergo a longer duration of phacoemulsification and higher phaco power, hence there is a four times higher risk of endothelial decompensation. Permanent endothelial decompensation is typically observed 4–6 weeks postoperatively, is a condition called bullous keratopathy. 7 The definitive management of bullous keratopathy is a corneal transplant. However, the ratio of donor availability and demand is 1:70. 8 There is a need for an alternative solution.

Total antioxidant capacity (TAC) portrays the capacity of a tissue to neutralize oxidants. As much as 73% of TAC in intraocular tissue is ascorbic acid (AA). 9 The amount of AA and TAC in the aqueous humor is found to be an independent protective factor towards endothelial decompensation. An increase of 1 mM TAC in aqueous humour causes 50 times lowered risk of endothelial decompensation. Protective features of AA towards oxidative stress in the cornea have been proven in vitro and in animal studies. In a rabbit corneal endothelial culture, topical AA is found to lower cell apoptosis. 10, 11 Using AA in irrigation solution in phacoemulsification for dogs is found to lower endothelial cell density, hexagonality, and variation coefficient. 12 On mild-to-moderate degree cataract, 500 mg of AA qid. lowers endothelial cell density difference to 30% compared to control. 13

Vitamin C has been widely studied for its potential to mitigate corneal endothelial damage caused by oxidative stress during phacoemulsification. Previous studies have demonstrated protective effects of vitamin C in mild to moderate cataracts using oral supplementation, as well as beneficial effects of topical administration in experimental animal models. 10, 11, 13 Oral vitamin C may represent a practical antioxidant due to its wide availability and lower risk of adverse effects compared with other antioxidant strategies such as intravenous vitamin C and intracameral antioxidants. Regarding these findings, we hypothesized that oral vitamin C supplementation increases ascorbic acid levels and overall antioxidant capacity in the aqueous humor and reduces oxidative stress–related endothelial damage during phacoemulsification. However, to date, no clinical studies have specifically evaluated the protective role of oral vitamin c supplementation in patients with hard cataracts undergoing phacoemulsification. On the other hand, aqueous humor will also be replaced by irrigation fluid during phacoemulsification, which may reduce the intraocular antioxidant content. Therefore, oral vitamin C is given postoperatively to restore AA levels in the aqueous humor. Thus, it is expected that the antioxidant effect of AA can suppress postoperative corneal endothelial autophagy and apoptosis, which can cause severe damage to the endothelial layer. Considering that aqueous humor sampling cannot be performed during the postoperative recovery period, it is important to determine whether the levels of MDA, TCA, and AA in the aqueous humor are the same as or correlate with blood levels, as no studies have yet examined these three biomolecular parameters simultaneously in both compartments.

 Protocol Methods Study design

This is a double-blinded, randomized controlled trial investigating the protective effect of ascorbic acid supplementation on corneal endothelial cells undergoing phacoemulsification. The study will follow the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) guidelines.

 Study setting

This study is going to be conducted in the Faculty of Medicine, University of Indonesia with samples from a regional hospital in Cianjur, West Java and Serang, Banten, Indonesia.

 Aims

The aim of this study is to analyze the protective effect of oral ascorbic acid supplementation on corneal endothelial cells which undergo oxidative stress due to phacoemulsification in patients with hard nucleus cataracts. The primary outcome will investigate mean change and percentage in endothelial cell density (ECD) loss prior to and after phacoemulsification, assessed at postoperative follow-up period (day 1, week 1, week 4, and week 6) of patients with hard nucleus cataracts.

The secondary endpoint will investigate mean change in other clinical parameters such as central corneal thickness (CCT), visual acuity, and cells in the anterior chamber during the postoperative follow-up period. This study will investigate differences in MDA levels in aqueous humor, prior to and after phacoemulsification, and compare between groups with vitamin C supplementation and those without. We will also investigate differences in AA, TAC, and MDA levels in serum and aqueous humor at the start of the study and after seven days of supplementation and compare them between the three groups. Furthermore, we will explore the correlation between aqueous humor and serum AA, TAC, and MDA levels.

 Sample size calculation

A total sampling method was used. The sample size was determined based on the primary outcome of postoperative corneal endothelial cell density (ECD) loss, referring to a previous study by Chee et al. 14 The minimal sample size calculated for this study was 36 subjects for each group (95% confidence interval, power 80%, β = 0.20, α = 0.05, SD = 201 ​​cell/mm 2, expected difference = 132 ​​cell/mm 2, subjects should have at least 132 ​​cell/mm 2 difference of ECD loss after phacoemulsification in the intervention group). With a 10% additional for anticipating loss to follow-up subjects, the minimal sample size needed was 40 for each group.

 Participants

The target population of this study is patients with hard nucleus cataracts who undergo phacoemulsification. Patients are eligible to be included in this study with the following inclusion criteria: aged above 60 years, have senile cataract in one or both eyes with Lens Opacities Classification System (LOCS) III nuclear opacity grade 4-6 and nuclear color grade 4-6, willing to consume prescription and participate in the study follow up for seven weeks starting from recruitment, and signed the informed consent. Written informed consent was obtained from all participants prior to enrollment in the study.

The exclusion criteria of this study are as follows: patients with vitamin C allergy or sensitivity, undergo intraoperative complications of postoperative infections, have corneal endothelium disorder, history of intraocular surgery, glaucoma, trauma, or other intraocular inflammations, have diabetes mellitus and kidney disorders, or consumes vitamins as daily supplements.

Samples will be dropped out during the study if they did not participate in the study follow-up or consume less than 80% of the prescribed medications.

 Screening

We screen patients who sign up for a mass cataract surgery event in a regional hospital in Cianjur, West Java, Indonesia. Patients will be asked for information regarding personal identification, history of systemic and ocular diseases, and allergies. They then will undergo ophthalmology examination including uncorrected visual acuity (UCVA) and best corrected visual acuity (BCVA), intraocular pressure using non-contact tonometry (NCT), biomicroscopy slit lamp, and indirect fundoscopy to evaluate anterior and posterior chambers. Lens opacity will then be measured using LOCS III criteria. We will also examine ECD, coefficient of variety (COV), and hexagonality of endothelial cell layers using a specular microscope (REM 4000, Rodenstock, Germany), central corneal thickness (CCT) using Anterior Segment Optical Coherence Tomography (ASOCT), and biometry. Blood samples will be then drawn from patients to measure serum AA, MDA, and TAC.

 Randomization, blinding and allocation concealment

The subjects will be allocated into three groups: Group 1 will receive oral vitamin C 500 mg, three times daily pre- and postoperatively, Group 2 will receive it only preoperatively, and Group 3 will receive a placebo. Participants will be randomly assigned to the study groups using a computer-generated block randomization sequence with a block size of four. The randomization sequence will be prepared by an independent researcher who will not be involved in patient recruitment or outcome assessment. Allocation concealment will be ensured using sequentially numbered, sealed, opaque envelopes. The study will be conducted in a double-blinded manner. Operating surgeons, examiners, laboratory analysts, and study participants will be blinded to group allocation throughout the study period. Participants will be followed up for seven weeks, and data will be analyzed after the minimum required number of participants has been reached. Figure 1 shows the planned CONSORT flow diagram of participants screening, randomization, follow-up, and analysis.

Planned CONSORT flow diagram of participants screening, randomization, follow-up, and analysis. Intervention

Patients will be divided into three groups, each with 40 samples. The first group (n=40) will receive 500 mg oral vitamin C three times daily (1500 mg/day) for one week before surgery to four weeks after surgery. The second group (n=40) will receive 500 mg oral vitamin C three times daily (1500 mg/day) for one week before surgery and a placebo after surgery. The last group (n=40) will receive a placebo one week before surgery to four weeks after surgery. Oral vitamin C 1500 mg/day was chosen based on pharmacokinetic evidence showing that this dosage can achieve stable plasma concentrations and approach a steady-state level during short-term administration. 15

To minimize potential bias related to surgical variability, all phacoemulsification procedures will be performed using standardized surgical techniques. The surgeries will be conducted by experienced cataract surgeons using the same phacoemulsification machine and standardized operative settings. Key intraoperative parameters, including ultrasound power, irrigation parameters, and the type of ophthalmic viscosurgical device (OVD), will be standardized across all procedures.

 Post-intervention evaluation

Patients will be asked to come a week after screening. Firstly, they asked the side effects of the supplementation, including gastrointestinal disorders (nausea, diarrhea, epigastric pain). Second blood samples will be then drawn to measure post-intervention serum AA, MDA, and TAC.

Sampling for oxidative stress in the aqueous humour will be performed twice, pre- and post-phacoemulsification. During surgery, after the initial incision, 0.2 mL of aqueous humor will be taken using a 1 cc syringe with a 30 G cannula. Phacoemulsification will then be conducted using standard operating procedure to intraocular lens implantation. Another 0.2 mL of aqueous humor will then be taken three minutes after removing the viscoelastic as a post-phacoemulsification sample. This sampling volume is within the range commonly used in aqueous humor biomarker studies and is considered safe under surgical conditions. Because the sampling will be performed through the surgical incision and the anterior chamber will subsequently be maintained with a balanced salt solution, anterior chamber stability will not be affected. 16, 17 The pre-phacoemulsification aqueous humour samples will be analysed for ascorbic acid (AA), total antioxidant capacity (TAC), and malondialdehyde (MDA), while the post-phacoemulsification samples will be analysed for MDA levels only. Intraoperative parameters, including effective phacoemulsification time (EPT) and total surgical duration, will be recorded for each procedure. All patients will receive topical medications: levofloxacin 5 mg/mL every three hours for one week, prednisolone acetate 10 mg/mL every three hours for 1 week, and tapered down in 4-6 weeks.

Every patient will be evaluated one day, one week, four weeks, and six weeks post-phacoemulsification to measure study parameters and signs of complications, i.e. inflammation or infection. Patients who experience side effects or complications, including toxic anterior segment syndrome (TASS) or endophthalmitis during the duration of follow-up will be exempted from the study and managed according to the standard operational procedure of the Department of Ophthalmology, Faculty of Medicine, University of Indonesia.

 Laboratory analysis

The examination of blood and aqueous humor samples will be conducted at the Pharmacology Laboratory, Faculty of Medicine, Universitas Indonesia (FMUI). Blood samples will be collected from all study participants who will have provided written informed consent. The collection, storage, and transportation procedures will be carried out under strict control of time and temperature to maintain sample stability. All participants will be gathered at the same time, and blood samples will be collected sequentially within a short period so that the entire process, from the first to the last sample, will be completed within one hour.

Immediately after collection, blood samples will be temporarily stored in a cooler box and will be transported from Sayang General Hospital, Cianjur, to the Pharmacology Laboratory, FMUI, under police escort from the Cianjur Police Department. The total transport time will be less than two hours. Centrifugation will be performed within six hours after collection at a speed of 1,000G for 15 minutes to obtain plasma, will be then stored at −80°C until further analysis.

Participants will be randomly assigned into three groups using computer-generated randomization: one placebo group and two intervention groups receiving oral vitamin C supplementation at predetermined doses. Each participant will be provided with a logbook containing the follow-up schedule, adverse effect monitoring, and contact information in case of any complaints. A second blood sample will be collected after seven days of supplementation for the analysis of ascorbic acid (AA), total antioxidant capacity (TAC), and malondialdehyde (MDA).

Aqueous humor samples will be collected twice, each of 0.2 mL: first, immediately after the incision (pre-phacoemulsification), and second, three minutes after viscoelastic removal following intraocular lens implantation (post-phacoemulsification). Both aqueous humor samples will be stored in a cool box containing dry ice at −78°C and will subsequently be transferred to long-term storage at −80°C in the Pharmacology Laboratory, FMUI, before analysis.

Principles of Analysis:

Measurement of Ascorbic Acid (AA) Levels

Ascorbic acid levels will be determined using an Enzyme-Linked Immunosorbent Assay (ELISA) Kit for Vitamin C (Cat. No. CEA913Ge). The principle of this assay is based on a competitive inhibition enzyme immunoassay method.

Measurement of Total Antioxidant Capacity (TAC)

Total antioxidant capacity will be measured using the QuantiChrom™ Antioxidant Assay Kit (DTAC-100, BioAssay Systems). This assay is based on the cupric ion reducing antioxidant capacity (CUPRAC) method.

Measurement of Malondialdehyde (MDA) Levels

MDA in Aqueous Humor:

Malondialdehyde levels in aqueous humor will be determined using a Malondialdehyde ELISA Kit (ab287797, Abcam). The principle of this assay is based on an indirect sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of MDA in serum, plasma, or other biological fluids.

MDA in Blood Samples:

Malondialdehyde levels in blood samples will be measured using a spectrophotometric method based on the reaction of MDA with thiobarbituric acid (TBA) to form a pink-colored complex that can be quantified spectrophotometrically.

Participant compensation

Participants receive IDR100,000 at the end of the last follow-up.

 Statistical analysis

Data from this study will be recorded and analyzed by IBM SPSS 20.0. Sample personal identification, including demographic and clinical characteristics, will be reported in a descriptive analysis. Data distribution of numerical variables will be assessed using the Shapiro–Wilk test with normal distribution will be presented as mean (standard deviation [SD]); non-normal distribution will be reported as median (interquartile range [IQR]). Nominal data will be reported in frequency and percentage. We will use Pearson’s chi-squared test for categorical data. For comparisons among the three groups, one-way ANOVA will be used for normally distributed data, while the Kruskal–Wallis test will be used for non-normally distributed data. Significant group differences will be followed by post-hoc pairwise comparisons using Bonferroni correction for ANOVA or Dunn’s test following the Kruskal–Wallis analysis to account for multiple testing. Pearson’s test will be used to test the correlation between data in aqueous humor and serum. A p-value of less than 0.05 (p < 0.05) will be considered statistically significant.

Effective phacoemulsification time (EPT) and total surgical duration will be included as covariates in the statistical analysis to account for their potential influence on postoperative endothelial outcomes. Multivariable analysis will also be conducted to control for potential confounding variables, including smoking status, occupational exposure, ultraviolet (UV) light exposure, and dietary intake of vitamin C. These variables will be collected through structured questionnaires during the screening.

Dissemination

The findings from this study will be disseminated through presentations at national and international conferences and submitted for publication in peer-reviewed journals. Trial outcomes will also be reported in the trial registry ( ClinicalTrials.gov). Authorship will follow ICMJE recommendations. The investigators intend to publish the findings regardless of the direction or significance of the results.

The results of this study will provide data on the protective effect of vitamin C supplementation for corneal endothelial cells in patients with hard nucleus cataracts undergoing phacoemulsification. This will initiate further investigation for the alternative management for permanent endothelial decompensation or bullous keratopathy.

Study status

At the time of manuscript submission (September 2025), the study is in the data processing phase. Trial registration has been completed ( ClinicalTrials.gov Identifier: NCT06781970).

Ethical considerations

This study was approved by the Institutional Review Board of the Faculty of Medicine, Universitas Indonesia – Cipto Mangunkusumo Hospital Ethics Committee with Approval number: KET-1252/UN2.F1/ETIK/PPM.00.02/2024 on 30 th August 2024. All participants provided written informed consent prior to participation.

 Data availability

No data are associated with this article as it describes a study protocol. Data generated from the study will be made openly available upon completion.

Underlying data

No underlying data are associated with this article as it describes a study protocol.

 Reporting guidelines

The completed SPIRIT 2013 checklist for this study is publicly available in Zenodo: SPIRIT checklist for “The Effects of Vitamin C Supplementation on Corneal Endothelial Damage in Hard Cataract Phacoemulsification: an Oxidative Stress Study on Aqueous Humour and Corneal Endothelial Cell Characteristics, A Randomized, Double-Blinded, Clinical Trial”. Zenodo. DOI: https://doi.org/10.5281/zenodo.17239356. 18

License: CC0 1.0 Universal.

 References Country Data : Indonesia [Internet]. IAPB Vision Atlas. [cited2026 Jan 29]. https://visionatlas.iapb.org/country-data/indonesia/ Girsang APLWH : Statistik penduduk lanjut usia 2021 [Internet]. Jakarta: Badan Pusat Statistik;2021[cited 2026 Jan 29]. pp.3–7. www.bps.go.id Davis G : The evolution of cataract surgery. Mo. Med. 2016;113(1):58–62. Bui AD Sun Z Wang Y : Factors impacting cumulative dissipated energy levels and postoperative visual acuity outcome in cataract surgery. BMC Ophthalmol. 2021;21(1):439. 34930170 10.1186/s12886-021-02203-3 PMC8690865 Tirzitis G Bartosz G : Determination of antiradical and antioxidant activity: Basic principles and new insights. Acta Biochim. Pol. 2010;57(2):139–142. Islam QU Saeed MK Mehboob MA : Age related changes in corneal morphological characteristics of healthy Pakistani eyes. Saudi J. Ophthalmol. 2017;31(2):86–90. 28559719 10.1016/j.sjopt.2017.02.009 PMC5436377 Khan MS Sharjeel M Ghaffar MT : Effect of phacoemulsification power on corneal endothelial cell count and corneal thickness in age related cataract surgery. Pak. J. Med. Health Sci. 2020;14(4):1261–1264. Reference Source Gain P Jullienne R He Z : Global survey of corneal transplantation and eye banking. JAMA Ophthalmol. 2016;134(2):167–173. 26633035 10.1001/jamaophthalmol.2015.4776 Tsao YT Wu WC Chen KJ : Analysis of aqueous humor total antioxidant capacity and its correlation with corneal endothelial health. Bioeng. Transl. Med. 2021;6(2):e10199. 34027088 10.1002/btm2.10199 PMC8126826 Hsueh Y-J Meir Y-JJ Yeh L-K : Topical ascorbic acid ameliorates oxidative stress-induced corneal endothelial damage via suppression of apoptosis and autophagic flux blockage. Cells. 2020;9(4):943. 32290365 10.3390/cells9040943 PMC7227019 Hsueh Y-J Meir Y-JJ Lai J-Y : Ascorbic acid ameliorates corneal endothelial dysfunction and enhances cell proliferation via the noncanonical GLUT1-ERK axis. Biomed. Pharmacother. 2021;144:112306. 34656060 10.1016/j.biopha.2021.112306 Padua IRM Valdetaro GP Lima TB : Effects of intracameral ascorbic acid on the corneal endothelium of dogs undergoing phacoemulsification. Vet. Ophthalmol. 2018;21(2):151–159. 28714237 10.1111/vop.12490 Bellarinatasari N Gunawan W Widayanti TW : The role of ascorbic acid on endothelial cell damage in phacoemulsification. Jurnal Oftalmologi Indonesia. 2011;7(5). Reference Source Chee S-P Yang Y Wong MHY . Randomized controlled trial comparing femtosecond laser-assisted with conventional phacoemulsification on dense cataracts. Am J Ophthalmol [Internet]. 2021;229:1–7. 10.1016/j.ajo.2020.12.024 Levine M Conry-Cantilena C Wang Y : Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3704–3709. 10.1073/pnas.93.8.3704 Xu W Liang Y Zhu Y Sun T Yuan Z Han X . Proteomic study of aqueous humour in diabetic patients with cataracts by TMT combined with HPLC-MS/MS. BMC Ophthalmol [Internet]. 2023;23(1). 10.1186/s12886-023-03162-2 Yin Z Gao Y Tang Y Tian X Zheng Y Han Q . Aqueous humor cytokine levels are associated with the severity of visual field defects in patients with primary open-angle glaucoma. BMC Ophthalmol [Internet]. 2023;23(1). 10.1186/s12886-023-02875-8 Widyawati S Sitompul R Wanandi SI : SPIRIT checklist for “The Effects of Vitamin C Supplementation on Corneal Endothelial Damage in Hard Cataract Phacoemulsification: an Oxidative Stress Study on Aqueous Humour and Corneal Endothelial Cell Characteristics, A Randomized, Double-Blinded, Clinical Trial” (Version 1) [Checklist]. Zenodo. 2025. 10.5281/zenodo.17239356 10.5256/f1000research.197958.r480069 Reviewer response for version 3 Tirsi Andrew 1 Referee https://orcid.org/0000-0002-8424-7574 Manhattan Eye, Ear, and Throat Hospital, Northwell Health, New York, USA

Competing interests: No competing interests were disclosed.

 15 5 2026 Copyright: © 2026 Tirsi A 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

This is not a research manuscript presenting results, and it does not look like a pilot study either.

The initial submission was 6 months ago and one would expect some preliminary data presented and discussed. Instead, this is only a study protocol, describing future steps of a study, with missing results, discussion and conclusion sections.

Please find some recommendations below:

1-The background is short, The last sentence is confusing, because as it is now, it describes the protective effects of AA in patients with hard nuclear cataracts (HNC). There is no mention of phacoemulsification (P). Therefore, the rationale of this research is missed. Phacoemulsification in HNC is very specific and authors should add in this work why specifically this type of cataracts is investigated here. Is it because the nucleus is very hard, high ultrasound power is used, more turbulence created during the procedure. Maybe this should be described in details to explain why this study is conducted.

2- Definitions should be added for HNC, and difficulties to make capsulorhesis when there is no red reflex, and other moments that make this procedure difficult and detrimental to the endothelial cells.

3-Introduction is missing references after every significant statement, risks for plagiarism 

4-The introduction is written in blocks, every paragraph not really linked to the previous one. not engaging the reader.

The gap of knowledge is not mentioned, and therefore the purpose of this study in not clear.

The last 3 lines in the introduction are not correct, because aqueous humor sampling can be done by aspiration (paracentesis) at the end of the surgery, and the use of experimental biosensors are used in research (ocucheck) that measures the electrical resistance of the aqueous humor for a quick quantitative analysis of vit c levels.

5-How was 7 days of vit c supplementation decided?

6-In the participants paragraph, please replace consume prescription by a better medical term

7-Why vit c was used in this study? and what was the advantage of oral supplementation? how about topical? 

8-will the side effects from the vit c supplementation monitored and registered?

8- please explain in details the aqueous humor sampling, safety justification and possible side effects. How is the second sample collected after 3 minutes after the surgery. Aren't the authors concerned about the volume of the saline used? this is going to be a cofounding factor, or diluting factor for the MDA levels. How to correct for this? Will Elisa results be affected by this dilution factor?

9- The manuscript initially was submitted in September 2025. I am sure authors could present some preliminary results to improve this manuscript in May 2026 (7-8 months)

Is the study design appropriate for the research question?

Partly

Is the rationale for, and objectives of, the study clearly described?

Partly

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

cataract surgery, glaucoma, glaucoma surgery

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.197958.r478734 Reviewer response for version 3 Chaurasiya Suraj Kumar 1 Referee https://orcid.org/0000-0002-7304-1714 Department of Optometry and Vision Science, CL Gupta Eye Institute, Moradabad, Uttar Pradesh, India

Competing interests: No competing interests were disclosed.

 5 5 2026 Copyright: © 2026 Chaurasiya SK 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work. The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions. recommendation approve

Thank you for considering the suggestion and making changes as per the comment. No further comment to be made.

Is the study design appropriate for the research question?

Partly

Is the rationale for, and objectives of, the study clearly described?

Partly

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Partly

Reviewer Expertise:

Contact Lens, Optometrist, Ocular disease, Optics, refractive surgery, Dry eyes

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.196193.r464837 Reviewer response for version 2 Chaurasiya Suraj Kumar 1 Referee https://orcid.org/0000-0002-7304-1714 Chaudhary Priyam 2 Co-referee Department of Optometry and Vision Science, CL Gupta Eye Institute, Moradabad, Uttar Pradesh, India CL Gupta Eye Institute, Moradabad, Uttar Pradesh, India

Competing interests: No competing interests were disclosed.

 12 3 2026 Copyright: © 2026 Chaurasiya SK and Chaudhary P 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

1. Knowledge Gap is Not Strongly Defined

The manuscript states that no clinical study has investigated oral vitamin C supplementation in hard nuclear cataracts, but the specific gap in the literature is not strongly emphasized.

The introduction should clearly state:

What is already known from previous antioxidant studies.

What remains unknown regarding systemic vitamin C supplementation and endothelial protection.

 2. The rationale does not adequately explain why oral vitamin C supplementation is chosen instead of other antioxidant strategies, such as:

topical antioxidants

Why 1500 mg/day was chosen.

intracameral antioxidants

antioxidant-enriched irrigation solutions.

Clarifying this would strengthen the translational relevance of the study.

 3. The manuscript would benefit from a clear hypothesis, for example:

Oral vitamin C supplementation increases intraocular antioxidant capacity and reduces oxidative stress–related endothelial damage during phacoemulsification.

4. The study includes 120 participants (40 per group), but no sample size calculation is provided.

Important details required:

Primary outcome used for power calculation.

Expected difference in endothelial cell density (ECD) loss.

Assumed standard deviation.

Alpha and power values.

 5. Without this information, the adequacy of the study design cannot be evaluated.

Although multiple outcomes are mentioned (ECD, CCT, visual acuity, inflammatory cells, oxidative stress markers), the primary endpoint is unclear.

The manuscript should clearly specify:

Primary outcome: e.g., percentage endothelial cell loss at 6 weeks

Secondary outcomes: CCT, oxidative stress markers, visual outcomes, postoperative inflammation.

6. This clarification is essential for RCT reporting.

The manuscript states that the trial is double-blinded, but details are lacking.

Required clarifications:

Who generated the randomization sequence?

Allocation concealment method (sealed envelopes, pharmacy randomization, etc.).

Who is blinded?

patients

surgeons

outcome assessors

laboratory investigators.

7. Without this information, the risk of bias cannot be assessed.

Endothelial cell loss during phacoemulsification strongly depends on intraoperative factors such as: cumulative dissipated energy (CDE), phaco time, ultrasound power, irrigation volume, viscoelastic type.

8. The protocol states that identical machines and consumables will be used, but intraoperative parameters should also be recorded and analyzed.

These should be included as covariates.

9. The protocol involves two aqueous humor samples during surgery.

Although small volumes (0.2 mL) are used, the manuscript should:

provide a safety justification

cite previous studies using similar sampling volumes

explain whether this affects anterior chamber stability.

 10. The statistical section needs refinement. Issues:

Multiple group comparison methods are not specified.

It is unclear whether ANOVA or Kruskal-Wallis tests will be used for the three-group comparison.

Adjustment for multiple comparisons should be described.

Multivariable analysis may be required to control for confounders such as:

age

nuclear grade

phaco energy.

11. "stress oxidative" Should be corrected to:  oxidative stress

12. Original: "Permanent endothelial decompensation usually observed 4–6 weeks postoperatively" Rewrite as: Permanent endothelial decompensation is typically observed 4–6 weeks postoperatively

13. The first group description states: "receive oral vitamin C, tid" The dose must be explicitly written as:  500 mg vitamin C three times daily (1500 mg/day)

14. The manuscript does not clearly state:  Institutional Review Board approval number and date of approval. These must be included.

Include a CONSORT flow diagram for screening, randomization, follow-up, analysis.

The protocol is interesting and potentially clinically impactful. However, clarification of the study design, statistical plan, and methodological details is necessary before publication.

Is the study design appropriate for the research question?

Partly

Is the rationale for, and objectives of, the study clearly described?

Partly

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Partly

Reviewer Expertise:

Contact Lens, Optometrist, Ocular disease, Optics, refractive surgery, Dry eyes

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Widyawati Syska Ophthalmology, University of Indonesia, Jakarta, DKI Jakarta, Indonesia

Competing interests: The authors declare that there are no competing interests regarding the publication of this article. We sincerely appreciate the reviewer’s constructive feedback, which has contributed to improving the clarity and overall quality of our manuscript.

 20 3 2026

We sincerely thank the reviewer for the positive evaluation of our protocol and for the constructive comments. We appreciate the recognition of its potential clinical impact. In response, we have carefully revised the manuscript to clarify the study design, statistical approach, and key methodological details as suggested.

1. We agree that the knowledge gap was not sufficiently highlighted in the original manuscript. The Introduction has been revised to better clarify the current evidence regarding antioxidant use in cataract surgery and to emphasize the remaining gap concerning systemic vitamin C supplementation and its potential protective effect on corneal endothelial cells, particularly in patients with hard nuclear cataracts. The revised paragraph now distinguishes what has been established in previous studies and what remains unclear, thereby strengthening the rationale for the present study.

2. We agree that the rationale for selecting oral vitamin C supplementation should be more clearly explained. The Introduction has been revised to clarify the advantages of systemic vitamin C administration compared with other antioxidant strategies. In particular, oral vitamin C supplementation may provide a practical and widely accessible approach with a lower risk of adverse effects that may enhance systemic and intraocular antioxidants. 

We have also clarified the rationale for selecting a dose of 1500 mg/day. Previous pharmacokinetic studies have shown that vitamin C supplementation at this dose can produce stable plasma concentrations and approach a steady-state level during short-term administration. This dosing strategy was selected to ensure adequate systemic antioxidant availability prior to surgery. This explanation has been added to the Introduction, and the dosing details are further described in the Intervention section.

3. We agree that clearly stating the study hypothesis would improve the clarity of the manuscript. Accordingly, a hypothesis statement has now been added at the end of the Introduction to better define the expected effects of oral vitamin C supplementation on intraocular antioxidant capacity and oxidative stress–related endothelial damage during phacoemulsification.

4. We agree that the sample size calculation should be clearly described. The Sample size calculation section has been revised to include details of the sample size estimation. The calculation was based on the primary outcome of postoperative corneal endothelial cell density (ECD) loss. The expected difference between groups and the assumed standard deviation were derived from previously published studies evaluating endothelial cell loss after phacoemulsification. A significance level (α) of 0.05 and a statistical power of 80% were used to determine the minimum sample size required for each group. The relevant details have now been added to the Sample size calculation section of the Methods.

5. We agree that the primary and secondary outcomes should be more clearly defined to improve the clarity of the study design. The Aim section of the Methods has been revised to explicitly state the primary outcome and secondary outcomes of the study. The primary outcome will be defined as the mean change and percentage change in corneal endothelial cell density (ECD) following phacoemulsification. Secondary outcomes will include other clinical parameters such as central corneal thickness (CCT), visual acuity, postoperative anterior chamber inflammation, as well as the mean differences and correlations of oxidative stress biomarkers measured in blood and aqueous humor. These clarifications have now been added to the Aim section of the Methods.

6. We agree that additional details regarding the randomization and blinding procedures are necessary to ensure transparency in reporting the randomized controlled trial. The Randomization, blinding and allocation concealment section of the Methods has been revised to clarify the process of randomization, allocation concealment, and blinding. Specifically, the randomization sequence will be generated using computer-based block randomization. Allocation concealment will be maintained using sequentially numbered, sealed, opaque envelopes that will be opened only after participant enrollment. Blinding will be maintained for operating surgeons, examiners, laboratory analysts, and study participants. These clarifications have now been added to the Randomization, blinding, and allocation concealment section of the Methods.

7. We agree that intraoperative factors may influence corneal endothelial cell loss during phacoemulsification and should be clearly described to reduce the potential risk of bias. The Intervention section has been revised to clarify that the surgical procedures will be performed using standardized techniques and equipment to minimize variability in intraoperative parameters. All surgeries will be conducted by experienced cataract surgeons using the same phacoemulsification machine and standardized surgical settings, including ultrasound power,  irrigation parameters, and the same type of ophthalmic viscosurgical device (OVD). These measures will help minimize procedural variability and reduce the influence of intraoperative factors on endothelial outcomes.

8. We agree that intraoperative factors may influence endothelial cell outcomes during phacoemulsification. To address this, the Methods section (post-intervention evaluation and statistical analysis section) has been revised to specify that intraoperative parameters, including effective phacoemulsification time (EPT) and total surgical duration, will be recorded for each procedure. These variables have now been added to  the statistical analysis as covariates to account for their potential influence on postoperative endothelial cell loss and other clinical outcomes.

9. We agree that clarification regarding the safety of aqueous humor sampling is necessary. The Post-intervention section of the Methods has been revised to explain that the volume of aqueous humor collected (0.2 mL) is within the range commonly used in ophthalmic research and is considered safe when performed under controlled surgical conditions. Previous studies investigating biochemical markers in aqueous humor during cataract surgery have also used similar sampling volumes without reported adverse effects. In addition, aqueous humor sampling will be performed twice, and because the sampling will be performed through the surgical incision and the anterior chamber will subsequently be maintained with balanced salt solution during the procedure, this approach will not compromise anterior chamber stability. Relevant references describing similar aqueous humor sampling volumes will be added to the manuscript.

10. The Statistical Analysis section has been revised to clarify the analytical approach used for comparisons among the three study groups. For normally distributed numerical variables, one-way analysis of variance (ANOVA) will be used, whereas the Kruskal–Wallis test will be applied for non-normally distributed data. When significant differences are detected, post-hoc pairwise comparisons with appropriate adjustment for multiple testing will be performed.

In addition, multivariable analysis will be conducted to account for potential confounding variables that may influence oxidative stress status and postoperative outcomes. These variables include smoking status, occupational exposure, ultraviolet (UV) light exposure, and dietary intake of vitamin C. These factors will be included in the analysis to minimize potential confounding effects on the association between vitamin C supplementation and study outcomes.

11. Thank you for pointing this out. The terminology has been corrected to “oxidative stress” throughout the manuscript.

12. Thank you for the suggestion. The sentence has been revised accordingly to improve grammatical clarity.

13. The dosage description has been revised to “500 mg vitamin C three times daily (1500 mg/day)” in the manuscript.

14. The Institutional Review Board approval number and date of approval have now been added to the Ethics Statement section of the manuscript. In addition, a CONSORT flow diagram illustrating participant screening, randomization, follow-up, and analysis has been included to improve the clarity of the study design and reporting. The flow diagram have now been added to the Randomization, blinding, and allocation concealment section of the Methods

We hope that these revisions have adequately addressed the reviewer’s concerns and have improved the clarity and rigor of the manuscript. We are grateful for the insightful feedback, which has significantly strengthened the quality of this work.

 10.5256/f1000research.196193.r456960 Reviewer response for version 2 Choudhary Rajesh 1 Referee Shri Shankaracharya College of Pharmaceutical Sciences, Bhilai, Chhattisgarh, India

Competing interests: No competing interests were disclosed.

 19 2 2026 Copyright: © 2026 Choudhary R 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

Authors addressed all my queries and suggestions.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Partly

Reviewer Expertise:

preclinical studies on animal for anticataract drugs

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.188354.r424633 Reviewer response for version 1 Choudhary Rajesh 1 Referee Shri Shankaracharya College of Pharmaceutical Sciences, Bhilai, Chhattisgarh, India

Competing interests: No competing interests were disclosed.

 5 11 2025 Copyright: © 2025 Choudhary R 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The study protocol was well designed. The study protocol lacks some details:

1. Level of significance not to mention: Before the statistical analysis of the data, significance level must be defined like P < 0.05 or P < 0.01

2. detailed procedure or citation not available for estimation of AA, MDA, and TCA. For reproducibility of the research, author must be clear defined the assay procedures, techniques, and methods used to measure the biochemical parameters.

3. Oxidative stress markers  are measured in serum only, while Phacoemulsification produces free radicals on eyes. so explain any justification or try to evaluate oxidative stress markers in aqueous humor also.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Partly

Reviewer Expertise:

preclinical studies on animal for anticataract drugs

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Widyawati Syska Ophthalmology, University of Indonesia, Jakarta, DKI Jakarta, Indonesia

Competing interests: The authors declare that there are no competing interests regarding the publication of this article. We sincerely appreciate the reviewer’s constructive feedback, which has contributed to improving the clarity and overall quality of our manuscript.

 6 11 2025

We sincerely thank the reviewer for the careful evaluation and valuable feedback on our study protocol. We appreciate these constructive suggestions, which have helped us improve the clarity and methodological rigor of our work.

1. Level of significance:

We agree with the reviewer and will specify the significance level for statistical analysis as P < 0.05. This will be added in the section “Statistical Analysis.”

2. Detailed procedure for estimation of AA, MDA, and TCA:

We appreciate this valuable suggestion. We will provide detailed information and appropriate citations for the methods used to estimate ascorbic acid (AA), malondialdehyde (MDA), and total antioxidant capacity (TCA). These details will be included in the revised version under “Laboratory Analysis.”

3. Oxidative stress markers in serum versus aqueous humour:

We thank the reviewer for this valuable comment. In fact, oxidative stress markers were also measured in the aqueous humour samples. Sampling was performed twice, pre- and post-phacoemulsification. The first sample (0.2 mL of aqueous humour) was collected immediately after corneal incision using a 1 cc syringe (Terumo) with a 30 G cannula. The second sample (0.2 mL) was obtained after intraocular lens implantation and viscoelastic removal, approximately 3 minutes later, using the same technique. The pre-phacoemulsification aqueous humour samples were analysed for ascorbic acid (AA), total antioxidant capacity (TAC), and malondialdehyde (MDA), while the post-phacoemulsification samples were analysed for MDA levels only. These details will be clarified and incorporated in the Post-intervention evaluation section of the revised version.

We sincerely thank the reviewer for the valuable and constructive feedback, which has strengthened the methodological transparency and scientific rationale of our research. We will prepare a revised version to address these points in detail.