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. 2025 Feb 27;20(2):e0319266. doi: 10.1371/journal.pone.0319266

Incidence of macular hole in patients undergoing pars plana vitrectomy for submacular hemorrhage

Riko Matsumoto 1,*, Masashi Kakinoki 1, Osamu Sawada 1, Shumpei Obata 1, Yoshitsugu Saishin 1, Masahito Ohji 1
Editor: Jiro Kogo2
PMCID: PMC11867310  PMID: 40014591

Abstract

Purpose

To investigate the incidence of macular hole (MH) during pars plana vitrectomy (PPV) for submacular hemorrhage (SMH) due to either retinal arterial macroaneurysm (RAM) rupture or age-related macular degeneration (AMD).

Methods

We retrospectively evaluated 47 eyes of 47 patients with SMH due to RAM rupture or AMD who underwent PPV. The presence or development of MHs was confirmed intraoperatively by the surgeon. We compared the incidence of MH between the RAM and AMD groups.

Results

In the RAM group, a MH was found in five of 28 (17.9%) eyes, and a MH was developed in three eyes by the surgical procedure. In the AMD group, there was no MH. All MHs were identified intraoperatively and closed by the same surgical procedure. In the RAM group, subretinal tissue plasminogen activator (t-PA) injection was performed in 12 eyes. Of the 12 eyes, two developed an MH before t-PA subretinal injection. Of the remaining 10 eyes, four (40%) developed an MH intraoperatively or postoperatively.

Conclusion

MHs are complications of PPV for SMH that occur more frequently in patients with SMH due to RAM rupture than in patients with SMH due to AMD.

Introduction

Submacular hemorrhage (SMH) is a condition that causes sudden vision loss and often results from retinal arterial macroaneurysm (RAM) rupture or age-related macular degeneration (AMD). Macular hemorrhages require rapid removal or displacement because they can produce irreversible iron- and fibrin-induced damage in outer layers of the retina [1,2]. Treatments for hemorrhage removal comprise pneumatic displacement with gas or pars plana vitrectomy (PPV). SMH can be treated by pneumatic displacement with gas or gas plus tissue plasminogen activator (t-PA) [3,4], but PPV with subretinal t-PA injection is preferred because it enables more reliable displacement. Furthermore, PPV is necessary in cases of SMH with sub-internal limiting membrane (ILM) hemorrhage or vitreous hemorrhage (VH).

Macular holes (MHs) reportedly occur in combination with SMH due to RAM rupture or AMD [5–8]. The presence of an MH is a factor associated with poor visual acuity after SMH displacement [9]. MHs can occur at various times: before surgery, during surgery, and after surgery [10]. To our knowledge, no studies have directly compared the incidence of MH due to SMH among underlying diseases. Therefore, this study retrospectively compared the incidence of MH in patients who underwent PPV for SMH between patients with SMH due to RAM rupture and patients with SMH due to AMD.

Patients and methods

This retrospective study included 47 patients with SMH due to RAM rupture or AMD who underwent PPV between April 2010 and June 2023 at the Shiga University of Medical Science Hospital. We excluded patients who had undergone pneumatic displacement of SMH before PPV. The study adhered to the tenets of the Declaration of Helsinki. The Shiga University of Medical Science Research Review Board approved the study protocol. Informed consent was obtained in the form of opt-out on the web-site. We accessed the data for research purposes in 25/08/2022-05/09/2022 and 27/08/2023. We have created anonymized information by giving research subjects research IDs that do not identify individuals on their own. The information obtained in the research is anonymized by managing it using the research ID.

Our indications for surgery in SMH are cases complicated by VH, preretinal hemorrhage or sub-ILM hemorrhage and cases where the size of the SMH is approximately one disc diameter or larger.

We retrospectively retrieved the following data from patients’ records: age, sex, best-corrected visual acuity (BCVA) (preoperatively and 1 month postoperatively), days from awareness of onset to surgery, preoperative VH, intraoperative t-PA use, ILM peeling involving the fovea, and Mention of MH in the surgical records. VH was determined based on fundus photographs and medical records. The presence or development of MH was confirmed by preoperative or postoperative optical coherence tomography (OCT) or intraoperatively by the surgeon. Surgeries were performed by five surgeons (M.O., M.K., O.S., H.K., and R.M.). The use of t-PA was determined at the surgeon’s discretion; t-PA was administered by preoperative intravitreal injection or by intraoperative subretinal injection. The surgical technique for subretinal injection of t-PA is as follows: the ILM at the planned t-PA injection site was peeled, and BSS was injected into the subretinal space using a micro cannula, after which 0.1 ml (25μg) of t-PA was injected. BSS was injected before tPA injection to inject the exact amount of tPA. The injection pressure was controlled using a viscous fluid control system (Alcon Laboratories, FortWorth, TX). ILM peeling was also performed at the surgeon’s discretion. All patients underwent fluid-air exchange at the end of surgery, followed by SF6 or air tamponade. Patients were instructed to remain in the prone position for 1–2 weeks postoperatively. We compared the incidence of MH in patients who underwent PPV for SMH between the RAM and AMD groups.

BCVA values measured by the Landolt C chart test were converted to logarithm of the minimum angle of resolution (logMAR) equivalents for statistical analysis. Based on a previous report, counting fingers was converted to a logMAR value of 1.85 and hand motion was converted to a logMAR value of 2.30 [11]. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) [12]. The Mann–Whitney U test was used to compare the RAM and AMD groups. Fisher’s exact test was used to compare the number of eyes with MH between the RAM and AMD groups. Data (age, logMAR BCVA, and days from onset) are expressed as means (standard deviations: SD). P values < 0.05 were considered statistically significant.

Results

This study included 28 eyes of 28 patients with SMH due to RAM rupture and 19 eyes of 19 patients with SMH due to AMD. The type of AMD was typical AMD in six eyes, polypoidal choroidal vasculopathy in twelve eyes, and retinal angiomatous proliferation in one eye. One patient in the RAM group and one patient in the AMD group were excluded from the study because they had undergone pneumatic displacement of SMH before PPV. The patients’ characteristics and surgical outcomes are presented in Table 1. The RAM group comprised 22 women and six men, whereas the AMD group comprised two women and 17 men (p < 0.01). The mean ages and standard deviations were 82.9 (SD 6.0) years (range, 67–91) in the RAM group and 73.2 (SD 8.5) years (range, 53–87) in the AMD group (p < 0.01). The mean times from onset to surgery were 9.6 (SD 7.4) days in the RAM group and 10.1 (SD 7.0) days in the AMD group; these did not significantly differ (p = 0.60). There were no significant differences in logMAR BCVA preoperatively or 1 month postoperatively between the two groups (preoperative: p = 0.06, postoperative: p = 0.55). Both groups showed significant improvement in BCVA after surgery (p < 0.01 for both analysis). Preoperative VH was observed in seven eyes (25.0%) in the RAM group and 10 eyes (52.6%) in the AMD group (p = 0.07). In the RAM group, intravitreal t-PA injection was performed in three eyes (10.7%); the day before surgery in two eyes, and during surgery in the other eye. In the AMD group, intravitreal t-PA injection was performed in two eyes (10.5%) the day before surgery. Subretinal t-PA injection was performed in 12 eyes (42.9%) in the RAM group and nine eyes (47.4%) in the AMD group (p = 0.77). ILM peeling at the central fovea was performed in 21 eyes (75.0%) in the RAM group and two eyes (10.5%) in the AMD group (p < 0.01). All data are provided in the S1 Dataset. The mean and median postoperative observation periods for the RAM group were found to be 13.3 and 12 months, respectively. The mean and median postoperative observation periods for the AMD group were found to be 40.9 and 37 months, respectively.

Table 1. Baseline characteristics and surgical outcomes.

RAM (n =  28) AMD (n =  19) P
Female/male 22/ 6 2/ 17  < 0.01
Age, years (SD) 82.9 (6.0) 73.2 (8.5)  < 0.01
Preoperative BCVA, logMAR (SD) 1.49 (0.40) 1.70 (0.54) 0.06
1M postoperative BCVA, logMAR (SD) 1.07 (0.48) 1.19 (0.55) 0.55
Days from onset (SD) 9.6 (7.2) 10.1 (7.0) 0.03
Preoperative vitreous hemorrhage (%) 7 (25.0) 10 (52.6) 0.07
Intravitreal t-PA injection, eyes (%) 3 (10.7) 2 (10.5) 1
Subretinal t-PA injection, eyes (%) 12 (42.9) 9 (47.4) 0.77
ILM peeling involving fovea, eyes (%) 21 (75.0) 2 (10.5)  < 0.01
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RAM: retinal artery macroaneurysm, AMD: age-related macular degeneration

BCVA: best-corrected visual acuity, ILM: internal limiting membrane.

In the RAM group, an MH was found in five of 28 eyes, and an MH was developed in three cases by the surgical procedure. The incidence of MH due to the RAM itself was 17.9%, and the incidence of MH related to the surgical procedure (subretinal t-PA injection) was 13.0%. In the AMD group, there was no MH (p < 0.01).

All MHs were identified intraoperatively and none were found by OCT. Table 2 shows baseline characteristics and surgical outcomes between the RAM patients who acquired and did not develop MH. ILM peeling involving the fovea was performed in six cases of the eight cases that developed MH. Subretinal t-PA injection was performed in four cases of these six cases. The times at which MHs were found or developed are presented in Fig 1. One MH was found immediately after core vitrectomy, but it was unclear whether the MH had occurred preoperatively or intraoperatively because the patient exhibited VH before the surgery. An MH was found during or immediately after ILM peeling in three eyes. An MH developed during intraoperative subretinal t-PA injection in three eyes. MH developed postoperatively in one case. MH of this case was found during a second surgery for rebleeding from the RAM. In the initial surgery of the eye, t-PA was injected into the subretinal space from a small area of ILM peeling outside the fovea. The following day, rebleeding from the RAM occurred, and a preretinal hemorrhage was observed in the macula. The second surgery was performed seven days after the air injected into the vitreous in the initial surgery had dissipated, and the preretinal hemorrhage was removed, revealing the MH. Therefore, the exact date of MH occurrence is unknown, but is considered to be within seven days. No OCT data on MH exist because sub-ILM hemorrhage or vitreous hemorrhage prevented OCT examination on MH and because all MH were found or developed intraoperatively and closed postoperatively.

Table 2. baseline characteristics and surgical outcomes between RAM patients who acquired and did not develop MH.

MH(+) (n =  8) MH(-) (n =  20) P
female/male 6/ 2 16/ 4 0.28
Age, years (SD) 80.8 (7.6) 83.8 (4.8) 0.51
Preoperative BCVA, logMAR (SD) 1.56 (0.39) 1.46 (0.40) 0.61
1M postoperative BCVA, logMAR (SD) 0.98 (0.51) 1.11 (0.47) 0.37
Days from onset (SD) 8.4 (2.8) 10.2 (8.3) 0.59
Preoperative vitreous hemorrhage (%) 2 (25.0) 5 (25.0) 1
Intravitreal t-PA injection, eyes (%) 0 3 (15.0) 0.54
Subretinal t-PA injection, eyes (%) 6 (75.0) 6 (30.0) 0.04
ILM peeling involving fovea, eyes (%) 6 (75.0) 15 (75.0) 1
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Fig 1. The times at which macular holes(MHs) were found or developed.

Fig 1

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MH was found immediately after core vitrectomy in one case. MH was found during or immediately after ILM peeling in three eyes. MH developed during intraoperative subretinal t-PA injection in three eyes. MH developed postoperatively in one case.

The MH was closed by same surgical treatment that identified MH in all eight cases. Five eyes were treated by ILM peeling and gas tamponade, whereas two eyes were treated using an inverted ILM flap technique and gas tamponade. In the remaining eye, a small extrafoveal region of the ILM was removed, and the MH developed during subretinal injection of balanced salt solution. In this eye, gas tamponade was performed without increasing the extent of ILM peeling. The mean logMAR BCVA values were 0.98 (SD 0.51) in eyes with MHs and 1.11 (SD 0.47) in eyes without MHs; these did not significantly differ (p = 0.37).

None of the patients who received intravitreal t-PA injection preoperatively or intraoperatively developed MH.

In the RAM group, subretinal t-PA injection was performed in 12 eyes. In two (16.7%) of the 12 eyes, an MH developed before subretinal t-PA injection. In four (40%) of the remaining 10 eyes, an MH developed intraoperatively or postoperatively. In eyes without subretinal t-PA injection, no MHs developed after ILM peeling.

Discussion

In this study, we compared 28 eyes with SMH due to RAM rupture and 19 eyes with SMH due to AMD. In this study, five MHs were found and three MHs developed intraoperatively in the RAM group. Eight of 28 eyes (28.6%) in the RAM group found or developed an MH, whereas no MHs occurred in the AMD group. There have been several reports of MHs in patients with SMH due to RAM rupture or SMH due to AMD [5–8,10,13–19], but no comparative studies have used the same methods at the same center. To our knowledge, this study is the first to directly compare the incidence of MH during or after vitrectomy between RAM and AMD groups at a single center.

Previous reports indicated that the incidences of MH associated with RAM rupture were 5-18% [18,20,21]. A previous meta-analysis revealed that the incidence of MH during PPV for RAM was 15% [22]. The reported incidences of MH in patients who underwent PPV for SMH due to RAM rupture were 9-50%. Although MH incidences vary among reports, they are expected to increase when study populations are limited to patients with SMH. In the present study, PPV was performed in patients with SMH due to RAM rupture; the total MH incidence was 8/28 (28.6%) and the MH incidence due to the RAM itself was 5/28 (17.9%), which are within the range of previous reports. In contrast, among previous reports concerning patients who underwent PPV for SMH due to AMD, MHs occurred in 1/36 (2.8%) [8] and 1/13 (7.7%) [17]. In the present study, there were no MHs among 19 patients. Published reports (including our study) indicate that the MH risk is much higher in eyes with SMH due to RAM rupture than in eyes with SMH due to AMD.

Several mechanisms have been proposed to explain the development of MHs in eyes with SMH. First, acute SMH might increase pressure in the subretinal space and lead to foveal dehiscence [19,20]. Second, tangential or anteroposterior vitreous traction into the fovea, due to VH-induced contraction of the posterior vitreous cortex, may contribute to MH development [5,7]. Third, persistent SMH causes retinal degeneration due to blood clot-derived fibrin, resulting in MH development [5,15,21]. Additionally, vitrectomy-induced mechanical damage to the macula can cause MH development [18,23]. If the timing of MH development was intraoperative or postoperative, the surgical procedures may have caused MH development. We speculate the main difference between RAM and AMD is the amount of subretinal pressure produced by the hemorrhage. The hemorrhage from RAM rupture originates from an artery and creates high subretinal pressure. The subretinal pressure generated by AMD-related hemorrhage is likely to be lower because this hemorrhage arises from choroidal neovascularization. Therefore, we suspect that subretinal pressure causes MHs more frequently in eyes with SMH due to RAM rupture than in eyes with SMH due to AMD.

Increased subretinal pressure caused by fluid injection may contribute to MH development. Our findings suggest that the incidence of MH is increased by subretinal t-PA injection for SMH due to RAM rupture. An MH developed during intraoperative subretinal t-PA injection in three eyes; an MH developed postoperatively in another eye that had undergone subretinal t-PA injection. Previous reports have described MH development in association with subretinal t-PA injection [16,18]. The previous findings are consistent with our results. MH has not occurred in patients with AMD who have undergone subretinal t-PA injection in this study. We consider that the risk of MH may be increased when subretinal t-PA infusion is performed in patients with RAM, because patients with SMH due to RAM have a weakened fovea, even if MH did not occur preoperatively. Although we speculate that subretinal fluid injection (including t-PA) increases the risk of MH development, it is difficult to draw a conclusion (based on this study alone) about whether subretinal t-PA injection should be used in surgical treatment of SMH due to RAM rupture; importantly, subretinal t-PA injection may improve SMH displacement and improve visual outcomes [24–26]. A larger prospective study is necessary to clearly establish the benefit of PPV with subretinal t-PA injection.

In this study, MHs were observed immediately after ILM peeling in three eyes. Even if an MH is not evident immediately after ILM peeling, the retina may become more fragile after ILM peeling, leading to increased incidence of MH. Kimura et al. reported a novel technique to prevent or treat MHs by creating a fissure near a sub-ILM hemorrhage outside of the central fovea and removing the sub-ILM hemorrhage from the fovea [27]. This technique may decrease the risk of MH development or may increase the likelihood of MH closure. Okanouchi et al. reported a technique for subretinal t-PA injection through a small area, in combination with ILM removal to decrease the injection pressure [28]. Although 12 psi was insufficient for subretinal t-PA injection into an area with intact ILM, subretinal injection through a small area was successfully performed using a pressure of 6 psi after ILM peeling. Subretinal injection with lower pressure might decrease the incidence of MH development, but this technique alone does not completely prevent MHs.

The present study is concerned with patients who have undergone PPV for SMH. To accurately assess the impact of PPV on MH, we decided to exclude patients who received other interventions before surgery; one patient in the RAM group and one patient in the AMD group received pneumatic treatment before surgery and were excluded from the study. The possibility cannot be excluded that this may have had an impact on the calculation of accurate MH incidence rates. This study may not accurately reflect the true incidence of the condition in all SMH patients, including those who were not scheduled for surgery.

In the present study, there was a relatively higher percentage of patients with VH in the AMD group (52.6%). Although pneumatic displacement with gas is often the preferred treatment for SMH due to AMD, PPV is required when VH is present. In our study, PPV was performed as initial treatment, so it is likely that a higher percentage of patients had VH.

There were several limitations in this study. First, it was a single-center, retrospective study with a small number of patients. Second, there were some differences in baseline characteristics (e.g., sex and age) between the RAM and AMD groups. Third, five surgeons participated in the study, and techniques such as ILM peeling or subretinal t-PA injection were performed at the surgeon’s discretion. A prospective study with larger cases is necessary to clearly establish the relationship between surgical techniques and MH development. Fourth, many patients had VH or preretinal hemorrhage before surgery, and the SMH size and retinal thickness before surgery were not considered.

In conclusion, this study showed that MHs more frequently occurred during or after PPV in eyes with SMH caused by RAM rupture than in eyes with SMH caused by AMD. The incidence of MH may increase due to subretinal fluid injection during surgical treatment of SMH caused by RAM rupture. Surgeons performing vitrectomy for SMH due to RAM rupture should be aware of the potential for MH development.

Supporting information

S1 Dataset. All data included in the analysis.

(XLSX)

pone.0319266.s001.xlsx (12.4KB, xlsx)

Acknowledgments

We thank Ryan Chastain-Gross, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

  • 1.Glatt H, Machemer R. Experimental subretinal hemorrhage in rabbits. Am J Ophthalmol. 1982;94(6):762–73. doi: 10.1016/0002-9394(82)90301-4 PMID: [DOI] [PubMed] [Google Scholar]
  • 2.Toth CA, Morse LS, Hjelmeland LM, Landers MB 3rd. Fibrin directs early retinal damage after experimental subretinal hemorrhage. Arch Ophthalmol. 1991;109(5):723–9. doi: 10.1001/archopht.1991.01080050139046 [DOI] [PubMed] [Google Scholar]
  • 3.Ohji M, Saito Y, Hayashi A, Lewis JM, Tano Y. Pneumatic displacement of subretinal hemorrhage without tissue plasminogen activator. Arch Ophthalmol. 1998;116(10):1326–32. doi: 10.1001/archopht.116.10.1326 [DOI] [PubMed] [Google Scholar]
  • 4.Fujikawa M, Sawada O, Miyake T, Kakinoki M, Sawada T, Kawamura H, et al. Comparison of pneumatic displacement for submacular hemorrhages with gas alone and gas plus tissue plasminogen activator. Retina. 2013;33(9):1908–14. doi: 10.1097/IAE.0b013e318287d99d [DOI] [PubMed] [Google Scholar]
  • 5.Colucciello M, Nachbar JG. Macular hole following ruptured retinal arterial macroaneurysm. Retina. 2000;20(1):94–6. doi: 10.1097/00006982-200001000-00018 [DOI] [PubMed] [Google Scholar]
  • 6.Mitamura Y, Terashima H, Takeuchi S. Macular hole formation following rupture of retinal arterial macroaneurysm. Retina. 2002;22(1):113–5. doi: 10.1097/00006982-200202000-00023 [DOI] [PubMed] [Google Scholar]
  • 7.Ciardella AP, Barile G, Schiff W, Del Priore L, Langton K, Chang S. Ruptured retinal arterial macroaneurysm associated with a stage IV macular hole. Am J Ophthalmol. 2003;135(6):907–9. doi: 10.1016/s0002-9394(02)02238-9 [DOI] [PubMed] [Google Scholar]
  • 8.Inoue N, Kato A, Araki T, Kimura T, Kinoshita T, Okamoto F, et al. Visual prognosis of submacular hemorrhage secondary to age-related macular degeneration: a retrospective multicenter survey. PLoS One. 2022;17(7):e0271447. doi: 10.1371/journal.pone.0271447 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Doi S, Kimura S, Saito S, Inoue M, Sakurai T, Kobori A, et al. Impact of macular intraretinal hemorrhage and macular hole on the visual prognosis of submacular hemorrhage due to retinal arterial macroaneurysm rupture. Retina. 2023;43(4):585–93. doi: 10.1097/IAE.0000000000003725 [DOI] [PubMed] [Google Scholar]
  • 10.van Zeeburg EJ, Cereda MG, van Meurs JC. Recombinant tissue plasminogen activator, vitrectomy, and gas for recent submacular hemorrhage displacement due to retinal macroaneurysm. Graefes Arch Clin Exp Ophthalmol. 2013;251(3):733–40. doi: 10.1007/s00417-012-2116-3 [DOI] [PubMed] [Google Scholar]
  • 11.Schulze-Bonsel K, Feltgen N, Burau H, Hansen L, Bach M. Visual acuities “hand motion” and “counting fingers” can be quantified with the freiburg visual acuity test. Invest Ophthalmol Vis Sci. 2006;47(3):1236–40. doi: 10.1167/iovs.05-0981 [DOI] [PubMed] [Google Scholar]
  • 12.Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48(3):452–8. doi: 10.1038/bmt.2012.244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Chino M, Yoshikawa Y, Kanno J, Nagashima T, Sakaki Y, Katsumoto T, et al. Development and spontaneous closure of a secondary macular hole associated with submacular hemorrhage due to polypoidal choroidal vasculopathy: a case report. BMC Ophthalmol. 2020;20(1):108. doi: 10.1186/s12886-020-01370-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lumi X, Drnovšek F. Management of a macular hole following retinal arterial macroaneurysm rupture. Am J Case Rep. 2020;21:e922437. doi: 10.12659/AJCR.922437 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.An JH, Jang JH. Macular hole formation following vitrectomy for ruptured retinal arterial macroaneurysm. Korean J Ophthalmol. 2021;35(3):248–50. doi: 10.3341/kjo.2020.1124 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Inoue M, Shiraga F, Shirakata Y, Morizane Y, Kimura S, Hirakata A. Subretinal injection of recombinant tissue plasminogen activator for submacular hemorrhage associated with ruptured retinal arterial macroaneurysm. Graefes Arch Clin Exp Ophthalmol. 2015;253(10):1663–9. doi: 10.1007/s00417-014-2861-6 [DOI] [PubMed] [Google Scholar]
  • 17.Kadonosono K, Arakawa A, Yamane S, Inoue M, Yamakawa T, Uchio E, et al. Displacement of submacular hemorrhages in age-related macular degeneration with subretinal tissue plasminogen activator and air. Ophthalmology. 2015;122(1):123–8. doi: 10.1016/j.ophtha.2014.07.027 [DOI] [PubMed] [Google Scholar]
  • 18.Nakamura H, Hayakawa K, Sawaguchi S, Gaja T, Nagamine N, Medoruma K. Visual outcome after vitreous, sub-internal limiting membrane, and/or submacular hemorrhage removal associated with ruptured retinal arterial macroaneurysms. Graefes Arch Clin Exp Ophthalmol. 2008;246(5):661–9. doi: 10.1007/s00417-007-0724-0 [DOI] [PubMed] [Google Scholar]
  • 19.Sato R, Yasukawa T, Hirano Y, Ogura Y. Early-onset macular holes following ruptured retinal arterial macroaneurysms. Graefes Arch Clin Exp Ophthalmol. 2008;246(12):1779–82. doi: 10.1007/s00417-008-0930-4 [DOI] [PubMed] [Google Scholar]
  • 20.Tashimo A, Mitamura Y, Ohtsuka K, Okushiba U, Imaizumi H, Takeda M. Macular hole formation following ruptured retinal arterial macroaneurysm. Am J Ophthalmol. 2003;135(4):487–92. doi: 10.1016/s0002-9394(02)02084-6 [DOI] [PubMed] [Google Scholar]
  • 21.Sagara N, Kawaji T, Koshiyama Y, Inomata Y, Fukushima M, Tanihara H. Macular hole formation after macular haemorrhage associated with rupture of retinal arterial macroaneurysm. Br J Ophthalmol. 2009;93(10):1337–40. doi: 10.1136/bjo.2008.149195 [DOI] [PubMed] [Google Scholar]
  • 22.Chen H, Zhao XY, Meng LH, Zhang WF, Chen YX. Clinical characteristics of retinal arterial macroaneurysms and prognosis of different interventions. Graefes Arch Clin Exp Ophthalmol. 2022;260(2):439–50. doi: 10.1007/s00417-021-05364-0 [DOI] [PubMed] [Google Scholar]
  • 23.Zhao P, Hayashi H, Oshima K, Nakagawa N, Ohsato M. Vitrectomy for macular hemorrhage associated with retinal arterial macroaneurysm. Ophthalmology. 2000;107(3):613–7. doi: 10.1016/s0161-6420(99)00127-x [DOI] [PubMed] [Google Scholar]
  • 24.Humayun M, Lewis H, Flynn HW, Sternberg P, Blumenkranz MS. Management of submacular hemorrhage associated with retinal arterial macroaneurysms. Am J Ophthalmol. 1998;126(3):358–61. doi: 10.1016/s0002-9394(98)00090-7 [DOI] [PubMed] [Google Scholar]
  • 25.Haupert CL, McCuen BW 2nd, Jaffe GJ, Steuer ER, Cox TA, Toth CA, et al. Pars plana vitrectomy, subretinal injection of tissue plasminogen activator, and fluid-gas exchange for displacement of thick submacular hemorrhage in age-related macular degeneration. Am J Ophthalmol. 2001;131(2):208–15. doi: 10.1016/s0002-9394(00)00734-0 [DOI] [PubMed] [Google Scholar]
  • 26.Wilkins CS, Mehta N, Wu CY, Barash A, Deobhakta AA, Rosen RB. Outcomes of pars plana vitrectomy with subretinal tissue plasminogen activator injection and pneumatic displacement of fovea-involving submacular haemorrhage. BMJ Open Ophthalmol. 2020;5(1):e000394. doi: 10.1136/bmjophth-2019-000394 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kimura A, Imai H, Iwane Y, Kishimoto M, Sotani Y, Yamada H, et al. Removal of sub-internal limiting membrane hemorrhage secondary to retinal arterial macroaneurysm rupture: internal limiting membrane non-peeling technique. J Clin Med. 2023;12(9):3291. doi: 10.3390/jcm12093291 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Okanouchi T, Toshima S, Kimura S, Morizane Y, Shiraga F. Novel technique for subretinal injection using local removal of the internal limiting membrane. Retina. 2016;36(5):1035–8. doi: 10.1097/IAE.0000000000001029 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Jiro Kogo

4 Oct 2024

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript raises interesting concern about the development of MH in patients who underwent PPV for SMH. The authors described the differences in features between individuals with RAM and AMD who had been selected for the surgery. However, there are certain points should be clarified in order to maximize the manuscript's usefulness.

Methods:

Nowadays, treatment options for SMH vary, including intravitreal gas injections with and without TPA, and with and without anti-VEGF factor injections. It would be interesting to know about the indications and ocular features of the individuals that led the physician to recommend PPV for this group of patients, such as the size or duration of SMH (if available). Without this information, reporting on the incidence of MH-associated SMH among these operated patients may be biased.

Results:

1. Differences in pre and intra-operative interventions, such as timing of TPA injection (pre vs intra-operative injection), route of TPA injection (intravitreal vs subretinal injection), and intra-operative ILM peeling (done vs not done), are all potential risk factors for MH development. As a result, I recommend to add more details of these methods (either in text or in table form), as seen below.

• In patients who received a pre-operative intravitreal TPA injection, how many cases developed MH?

• In patients with ILM peeling and experienced MH, how many cases received subretinal gas/TPA injections?

• In patients that developed post-operative MH, how many days after surgery did it develop?

2. Regarding MH, it is interesting to know the difference in baseline features between RAM patients who acquired and did not develop MH. The information provided may be beneficial to the readers. I recommend that authors incorporate this information.

Discussion:

1. The authors should clarify that the incidence of MH in PPV patients may not accurately reflect the true incidence in all SMH patients which including those who did not schedule the operation.

2. The authors should add and emphasize that patients with pre-operative MH may not have the same mechanism as those who developed MH during or after vitrectomy, which could be due to disease pathology or surgical maneuvers.

3. 3. In the discussion of subretinal gas/TPA injection and MH, please further note that the volume and pressure might vary between patients due to retrospective studies. As a result, the relationship between subretinal TPA injection and MH should be investigated further in a prospective design.

Table 1:

Correct row for "preoperative VH"

Reviewer #2: I had the pleasure to review the manuscript by Matsumoto et al investigating the incidence of macular hole (MH) during pars plana vitrectomy for submacular hemorrhage due to either retinal arterial microaneurysm rupture or age-related macular degeneration. The manuscript is well formatted and fluent. The study design is clear and the introduction is well focused. The methods are widely explained and the results are clearly analyzed. The discussion is fluent , updated and solid.

Reviewer #3: Dear authors there appears to be a conflict trying to understand if the manuscript is focused on MH as a complication of SMH surgery or MH occuring after SMH in which case surgery (PPV) is not to be consider. You need to resolve this conflict or modify your title . Please se my comments below to provide some more information on your manuscript

Title : The title raises questions. First it gives the impression that the MH developed as a result of the surgery. But this is not the case as some eyes had MH even before the surgery, while other eyes developed the MH during or after the surgery. So this is a mixed group of MHs all related to the SMH and not necessarily to the surgery. The author needs to decide on if the surgery for SMH or the SMH is the focus of research.

Question: Were all the macular holes formed or noticed during vitreoretinal surgery? What of MHs that occurred post vitrectomy? How long after the primary surgery did the post operative MH occur? Were there extrafoveal MHs too?

The abstract’s purpose states that the MHs was during PPV for SMH. This does not appear to be correct and reflective of the work done as pre operative and post operative OCTs reflects timing of the MH as not only during surgery.

What does the author mean by” initial surgical treatment”?

What was the indication for the surgery and was present vitreous hemorrhage an indication for surgery in some cases? What technique was used for the surgery? Was it submacular surgery with subretinal tPA? Then a detailed description of the surgical procedure is required including the site of subretinal tPA injection.

Please include the mechanism of MH formation in the eyes with SMH?

Since this was a retrospective study, was there a standardized surgical procedure for all the cases.

Introduction section:

Again in line 47, “patients who underwent PPV” appears to mean that the focus is on the PPV for SMH.

Methods section:

Line 53: Why were patients who underwent pneumatic displacement before PPV excluded? This has a direct effect on the incidence. How many were excluded?

Line 63: Incidence of MH would not be one of the data extracted from the patients’ records. Rather MH and its dimensions on OCT would be a more likely data extracted.

Line 80: Which data was expressed as means and standard deviation?

Results section:

In table 1, the proportion of eyes with VH in the RAM group is missing.

I would have liked to see the OCT based size of the MHs including the minimum or basal diameter. I appreciate that this may not be available for all eyes since some eyes had a media opacity including VH.

It appears that subretinal t-PA carried a risk for the formation of intraoperative MH? Can the authors perform an analysis to examine the risk element comparing t-PA with the other grous.

Also, I would imagine that the height of the SMH would contribute to the mechanical stretching of the macular and cause considerable tangential and posterior anterior traction that could predispose to the development of the MH. Therefore can the author give an idea of the OCT based height of the SMH between the RAM and AMD eyes. More SMH and more height of elevated retina would be associated with more MH.

How many MHs occurred after vitrectomy? What was the time from vitrectomy to the occurrence of the MHs?

What was the rate of follow in the two groups?

A major drawback of this study is that multiple surgical techniques by multiple surgeons is likely to have been employed as this is a retrospective study. So it is truly difficult to compare and draw a reasonable conclusion since the surgical techniques are not controlled for. This general is a difficult study like any other surgical research in which the surgical techniques and steps are not controlled.

Discussion:

Because of the excluded eyes, the incidence is likely to be under estimated. Please provided the number of eyes excluded

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:  OGUGUA NDUBUISI OKONKWO

**********

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PLoS One. 2025 Feb 27;20(2):e0319266. doi: 10.1371/journal.pone.0319266.r003

Author response to Decision Letter 1

6 Nov 2024

Dear Dr. Kogo, and Reviewers,

Manuscript ID PONE-D-24-29059

Title: Incidence of macular hole in patients undergoing pars plana vitrectomy for submacular hemorrhage

Thank you very much for your careful review that contained important suggestions to improve our manuscript. We have studied the comments carefully and have made corrections, which we hope meet your approval.

Reviewer #1:

Methods: Nowadays, treatment options for SMH vary, including intravitreal gas injections with and without TPA, and with and without anti-VEGF factor injections. It would be interesting to know about the indications and ocular features of the individuals that led the physician to recommend PPV for this group of patients, such as the size or duration of SMH (if available). Without this information, reporting on the incidence of MH-associated SMH among these operated patients may be biased.

Our indications for surgery in SMH are cases complicated by VH, preretinal hemorrhage or sub-ILM hemorrhage and cases where the size of the SMH is approximately one disc diameter or larger. This statement has been added to Patients and Methods (page4, lines 59-61).

Results:

1. Differences in pre and intra-operative interventions, such as timing of TPA injection (pre vs intra-operative injection), route of TPA injection (intravitreal vs subretinal injection), and intra-operative ILM peeling (done vs not done), are all potential risk factors for MH development. As a result, I recommend to add more details of these methods (either in text or in table form), as seen below.

• In patients who received a pre-operative intravitreal TPA injection, how many cases developed MH?

The number of patients who received intravitreal t-PA injection was added to the results and table1. No cases developed MH (page 6, lines 105-107).

• In patients with ILM peeling and experienced MH, how many cases received subretinal gas/TPA injections?

ILM peeling involving the fovea was performed in six cases of the eight cases that developed MH. Subretinal t-PA injection was performed in four cases of these six cases. (page7, lines123-125).

• In patients that developed post-operative MH, how many days after surgery did it develop?

MH developed postoperatively in one case. MH of this case was found during a second surgery for rebleeding from the RAM. In the initial surgery of the eye, t-PA was injected into the subretinal space from a small area of ILM peeling outside the fovea. The following day, rebleeding from the RAM occurred, and a preretinal hemorrhage was observed in the macula. The second surgery was performed seven days after the air injected into the vitreous in the initial surgery had dissipated, and the preretinal hemorrhage was removed, revealing the MH. Therefore, the exact date of MH occurrence is unknown, but is considered to be within seven days. (page 7, line 129-136).

2. Regarding MH, it is interesting to know the difference in baseline features between RAM patients who acquired and did not develop MH. The information provided may be beneficial to the readers. I recommend that authors incorporate this information.

In accordance with the reviewer's comment, we have added Table2 (page8).

Discussion:

1. The authors should clarify that the incidence of MH in PPV patients may not accurately reflect the true incidence in all SMH patients which including those who did not schedule the operation.

In accordance with the reviewer's comment, we have added the following sentence into the Discussion. “This study may not accurately reflect the true incidence of the condition in all SMH patients, including those who were not scheduled for surgery.” (page11, line 221-222)

2. The authors should add and emphasize that patients with pre-operative MH may not have the same mechanism as those who developed MH during or after vitrectomy, which could be due to disease pathology or surgical maneuvers.

In accordance with the reviewer's comment, we have added the following sentence into the Discussion. “If the timing of MH development was intraoperative or postoperative, the surgical procedures may have caused MH development.” (page 10, line 185-186)

3. In the discussion of subretinal gas/TPA injection and MH, please further note that the volume and pressure might vary between patients due to retrospective studies. As a result, the relationship between subretinal TPA injection and MH should be investigated further in a prospective design.

In accordance with the reviewer's comment, we have added the following sentence into the Discussion. “A prospective study with larger cases is necessary to clearly establish the relationship between surgical techniques and MH development.” (page 12, line 231-232)

Table 1: Correct row for "preoperative VH"

We corrected this.

Reviewer #2:

I had the pleasure to review the manuscript by Matsumoto et al investigating the incidence of macular hole (MH) during pars plana vitrectomy for submacular hemorrhage due to either retinal arterial microaneurysm rupture or age-related macular degeneration. The manuscript is well formatted and fluent. The study design is clear and the introduction is well focused. The methods are widely explained and the results are clearly analyzed. The discussion is fluent, updated and solid.

We greatly appreciate your comment.

Reviewer #3

Dear authors there appears to be a conflict trying to understand if the manuscript is focused on MH as a complication of SMH surgery or MH occuring after SMH in which case surgery (PPV) is not to be consider. You need to resolve this conflict or modify your title. Please my comments below to provide some more information on your manuscript

Title : The title raises questions. First it gives the impression that the MH developed as a result of the surgery. But this is not the case as some eyes had MH even before the surgery, while other eyes developed the MH during or after the surgery. So this is a mixed group of MHs all related to the SMH and not necessarily to the surgery. The author needs to decide on if the surgery for SMH or the SMH is the focus of research.

All MHs that occurred in this study were discovered intraoperatively; we emphasize that all MHs were discovered intraoperatively to make it clear that the purpose of this study was to examine the impact of surgery on SMH. (page 7, line 121)

Question: Were all the macular holes formed or noticed during vitreoretinal surgery? What of MHs that occurred post vitrectomy? How long after the primary surgery did the post operative MH occur? Were there extrafoveal MHs too?

All MHs were found or developed during surgery. MH developed postoperatively in one case. MH of this case was found during a second surgery for rebleeding from the RAM. In the initial surgery of the eye, t-PA was injected into the subretinal space from a small area of ILM peeling outside the fovea. The following day, rebleeding from the RAM occurred, and a preretinal hemorrhage was observed in the macula. The second surgery was performed seven days after the air injected into the vitreous in the initial surgery had dissipated, and the preretinal hemorrhage was removed, revealing the MH. Therefore, the exact date of MH occurrence is unknown, but is considered to be within seven days. (page 7, line 129-136). There was no extrafoveal MHs.

The abstract’s purpose states that the MHs was during PPV for SMH. This does not appear to be correct and reflective of the work done as pre operative and post operative OCTs reflects timing of the MH as not only during surgery.

In this study, all MHs were found or developed during surgery. The phrase “by preoperative or postoperative optical coherence tomography” was removed from the abstract.

What does the author mean by” initial surgical treatment”?

“Initial surgical treatment” refers to the first surgery if the patient has had multiple surgeries.

What was the indication for the surgery and was present vitreous hemorrhage an indication for surgery in some cases?

This includes cases in which SMH was found during surgery because fundus examination could not be performed before surgery due to vitreous hemorrhage.

What technique was used for the surgery? Was it submacular surgery with subretinal tPA? Then a detailed description of the surgical procedure is required including the site of subretinal tPA injection.

We have added a description of the surgical technique to the METHODS section. The surgical technique for subretinal injection of t-PA is as follows: the ILM at the planned t-PA injection site was peeled, and BSS was injected into the subretinal space using a micro cannula, after which 0.1 ml (25μg) of t-PA was injected. BSS was injected before tPA injection to inject the exact amount of tPA. The injection pressure was controlled using a viscous fluid control system (Alcon Laboratories, FortWorth, TX). (page 4, line 70-74)

Please include the mechanism of MH formation in the eyes with SMH?

The mechanism of MH formation in SMH is described in Discussion. (page 10, line179-186)

Since this was a retrospective study, was there a standardized surgical procedure for all the cases.

Since this is a retrospective study and the duration of the study is as long as 13 years, there is no standardized surgical procedure for all cases. The surgical procedure was determined at the discretion of the surgeons, and this is noted in the limitations. (page 12 line 229-23)

Introduction section:

Again in line 47, “patients who underwent PPV” appears to mean that the focus is on the PPV for SMH.

This study focuses on patients who received PPV for SMH.

Line 53: Why were patients who underwent pneumatic displacement before PPV excluded? This has a direct effect on the incidence. How many were excluded?

To accurately assess the impact of PPV on MH, we decided to exclude patients who received other interventions before surgery; one patient in the RAM group and one patient in the AMD group received pneumatic treatment before surgery and were excluded from the study. (page 11, line 216-222)

Line 63: Incidence of MH would not be one of the data extracted from the patients’ records. Rather MH and its dimensions on OCT would be a more likely data extracted.

The phrase “incidence of MH” was changed to “Mention of MH in the surgical records” in response to the reviewer's comment (page 4, line 65). No OCT data on MH exist because sub-ILM hemorrhage or vitreous hemorrhage prevented OCT examination on MH and because all MH were found or developed intraoperatively and closed postoperatively.

Line 80: Which data was expressed as means and standard deviation?

In accordance with the reviewer's comment, we have added to the Methods which data it is.

Results section:

In table 1, the proportion of eyes with VH in the RAM group is missing.

We corrected this.

I would have liked to see the OCT based size of the MHs including the minimum or basal diameter. I appreciate that this may not be available for all eyes since some eyes had a media opacity including VH.

No OCT data on MH exist because all MH were found or developed intraoperatively and closed postoperatively.

It appears that subretinal t-PA carried a risk for the formation of intraoperative MH? Can the authors perform an analysis to examine the risk element comparing t-PA with the other grous.

As the reviewer has noted, there is a potential risk of intraoperative MH formation associated with subretinal injection of t-PA. However, it is regrettable that further analysis cannot be conducted in the present study.

Also, I would imagine that the height of the SMH would contribute to the mechanical stretching of the macular and cause considerable tangential and posterior anterior traction that could predispose to the development of the MH. Therefore can the author give an idea of the OCT based height of the SMH between the RAM and AMD eyes. More SMH and more height of elevated retina would be associated with more MH.

As the reviewer asserted, we concur that the height of SMH based on OCT is a valuable indicator for investigating the predisposition for the development of MH. However, in the present study, the preoperative SMH height could be assessed in only a few cases due to the presence of sub-ILM hemorrhage or VH. Only 11 of the 28 cases in the RAM group and 3 of the 19 cases in the AMD group had SMH height that was measurable by OCT. Consequently, the paper did not include information on SMH height by OCT.

How many MHs occurred after vitrectomy? What was the time from vitrectomy to the occurrence of the MHs?

One MH occurred after vitrectomy. MH of this case was found during a second surgery for rebleeding from the RAM. In the initial surgery of the eye, t-PA was injected into the subretinal space from a small area of ILM peeling outside the fovea. The following day, rebleeding from the RAM occurred, and a preretinal hemorrhage was observed in the macula. The second surgery was performed seven days after the air injected into the vitreous in the initial surgery had dissipated, and the preretinal hemorrhage was removed, revealing the MH. (page 7, line 129-136)

What was the rate of follow in the two groups?

The mean and median postoperative observation periods for the RAM group were found to be 13.3 and 12 months, respectively. The mean and median postoperative observation periods for the AMD group were found to be 40.9 and 37 months, respectively. (page 6, line 110-113)

A major drawback of this study is that multiple surgical techniques by multiple surgeons is likely to have been employed as this is a retrospective study. So it is truly difficult to compare and draw a reasonable conclusion since the surgical techniques are not controlled for. This general is a difficult study like any other surgical research in which the surgical techniques and steps are not controlled.

As the reviewer pointed out, one of the shortcomings of this study is the lack of control over surgical techniques and procedures. We have added the following language to our discussion:” A prospective study with larger number of cases is necessary to clearly establish the relationship between surgical techniques and MH.”

Discussion:

Because of the excluded eyes, the incidence is likely to be under estimated. Please provided the number of eyes excluded

One patient in the RAM group and one patient in the AMD group received pneumatic treatment before surgery and were excluded from the study. The possibility cannot be excluded that this may have had an impact on the calculation of accurate MH incidence rates. We have added this to our results and discussion. (page 5 line 93-95, page 11 line 216-222)

We greatly appreciate both your help and that of the referees concerning improvements to this paper. I hope that the revised manuscript is now suitable for publication.

Sincerely yours,

Riko Matsumoto, MD, corresponding author

Department of Ophthalmology, Shiga University of Medical Science

Seta-Tsukinowacho, Otsu, Shiga 520-2192, JAPAN

Tel: +81-77-548-2276

Fax: +81-77-548-2279

e-mail: rokamoto@belle.shiga-med.ac.jp

Attachment

Submitted filename: Response to Reviewers.docx

pone.0319266.s003.docx (27.4KB, docx)

Decision Letter 1

Jiro Kogo

29 Nov 2024

PONE-D-24-29059R1Incidence of macular hole in patients undergoing pars plana vitrectomy for submacular hemorrhagePLOS ONE

Dear Dr. Matsumoto,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 13 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Jiro Kogo

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I would like to express my gratitude to the authors for their substantial contribution to the scientific knowledge conveyed in this study. Additionally, the limitations related to the retrospective data analysis have been emphasized more clearly. I have a few minor recommendations for the authors.

Introduction and abstract and Table:

- It is advisable to use the genera term "rupture arterial macroaneurysm" in place of " rupture arterial microaneurysm."

Results:

� Line: 105-107

“Intravitreal t-PA injection was performed in three eyes (10.7%, day before surgery: 2, in surgery: 1) in the RAM group and two eyes (10.5%, 107 before surgery: 2) in the AMD group (p=1).”

� This statement presents challenges in interpretation. It is advisable to eliminate either surgery 1 or surgery 2 from this statement and describe using an alternative sentence.

� Line 155:

“None of the patients who received intravitreal t-PA injection developed MH.”

� It would be more accurate to state the time point of injection, (preoperative, correct?).

Table 1 and Table 2:

� Considering use:

“Preoperative intravitreal t-PA” instead of “intravitreal t-PA”.

“Intraoperative subretinal t-PA injection” instead of “subretinal t-PA injection”

It is advisable to include the Mean ± SD within parentheses for continuous variables.

Reviewer #3: Dear Authors, it is my impression that you have put in a lot of work to revise this manuscript. However, I still have difficulties getting a clear understanding of the reporting of your research.

For example, you have stated that all the macular holes were discovered intraoperatively, this is not clearly reflected in the abstract.

Please state clearly that in all cases the MH was “discovered” during PPV after removal of the vitreous hemorrhage. This helps explain why the MH was identified only during PPV.

Secondly, following your explanation in the revised letter, the incidence can not include those cases in which MH was induced by the surgical procedure or subretinal TPA injection. To give the true incidence, the sample MUST be representative of only those cases that was as a result of RAP or nAMD only and not iatrogenic. Therefore those MHs that developed following surgery have to be excluded all through the manuscript.

“Purpose: To investigate the incidence of macular hole (MH) during pars plana vitrectomy (PPV) for submacular hemorrhage (SMH) due to either retinal arterial microaneurysm (RAM) rupture or age-related macular degeneration (AMD). Methods: We retrospectively evaluated 47 eyes of 47 patients with SMH due to RAM rupture or AMD who underwent PPV. The presence or development of MHs was confirmed intraoperatively by the surgeon. We compared the incidence of MH between the RAM and AMD groups

This understanding is also lacking in the later segment below

“The MH was closed by initial surgical treatment in all eight cases. In the RAM group, subretinal tissue plasminogen activator (t-PA) injection was performed in 12 eyes. Of the 12 eyes, two developed an MH before t-PA subretinal injection. Of the remaining 10 eyes, four (40%) developed an MH intraoperatively or postoperatively”

First, please make it clear that the MH was closed during the same procedure to remove the vitreous hemorrhage. Initial surgical treatment gives the impression that a planned MH surgery was done.

Secondly, those eyes that developed MH after t-PA injection should be excluded for the simple reason that the intervention could be the reason for MH and not entirely the disease. So, if the injection was not done, the MH may not have occurred. This point has to be reflected in the manuscript.

**********

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Reviewer #1: No

Reviewer #3: Yes:  OGUGUA NDUBUISI OKONKWO

**********

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PLoS One. 2025 Feb 27;20(2):e0319266. doi: 10.1371/journal.pone.0319266.r005

Author response to Decision Letter 2

8 Jan 2025

Response to Reviewers letter

Dr. Jiro Kogo

Academic Editor, PLOS ONE

Dear Dr. Kogo, and Reviewers,

Manuscript ID PONE-D-24-29059

Title: Incidence of macular hole in patients undergoing pars plana vitrectomy for submacular hemorrhage

Thank you very much for your careful review that contained important suggestions to improve our manuscript. We have studied the comments carefully and have made corrections, which we hope meet your approval.

Reviewers’ comments are reproduced and are followed by our responses.

Reviewer #1

I would like to express my gratitude to the authors for their substantial contribution to the scientific knowledge conveyed in this study. Additionally, the limitations related to the retrospective data analysis have been emphasized more clearly. I have a few minor recommendations for the authors.

Introduction and abstract and Table:

- It is advisable to use the genera term "rupture arterial macroaneurysm" in place of " rupture arterial microaneurysm."

We corrected this term.

Results:

� Line: 105-107

“Intravitreal t-PA injection was performed in three eyes (10.7%, day before surgery: 2, in surgery: 1) in the RAM group and two eyes (10.5%, 107 before surgery: 2) in the AMD group (p=1).”

� This statement presents challenges in interpretation. It is advisable to eliminate either surgery 1 or surgery 2 from this statement and describe using an alternative sentence.

The sentence pointed out (Line: 105-107) was eliminated, and the following sentences were added. “In the RAM group, intravitreal t-PA injection was performed in three eyes (10.7%); the day before surgery in two eyes, and during surgery in the other eye. In the AMD group, intravitreal t-PA injection was performed in two eyes (10.5%) the day before surgery.”

� Line 155:

“None of the patients who received intravitreal t-PA injection developed MH.”

� It would be more accurate to state the time point of injection, (preoperative, correct?).

We have added the phrase “preoperatively or intraoperatively” to the sentence. (page9, line159)

Table 1 and Table 2:

� Considering use:

“Preoperative intravitreal t-PA” instead of “intravitreal t-PA”.

“Intraoperative subretinal t-PA injection” instead of “subretinal t-PA injection”

We could not make this change because “intravitreal t-PA injection” is included both intraoperatively and preoperatively.

It is advisable to include the Mean ± SD within parentheses for continuous variables.

In accordance with the reviewer's comment, the notation of the standard deviation has been changed.

Reviewer #3

Dear Authors, it is my impression that you have put in a lot of work to revise this manuscript. However, I still have difficulties getting a clear understanding of the reporting of your research.

For example, you have stated that all the macular holes were discovered intraoperatively, this is not clearly reflected in the abstract.

Please state clearly that in all cases the MH was “discovered” during PPV after removal of the vitreous hemorrhage. This helps explain why the MH was identified only during PPV.

We added in the abstract that all MH were found intraoperatively.

Secondly, following your explanation in the revised letter, the incidence can not include those cases in which MH was induced by the surgical procedure or subretinal TPA injection. To give the true incidence, the sample MUST be representative of only those cases that was as a result of RAP or nAMD only and not iatrogenic. Therefore those MHs that developed following surgery have to be excluded all through the manuscript.

“Purpose: To investigate the incidence of macular hole (MH) during pars plana vitrectomy (PPV) for submacular hemorrhage (SMH) due to either retinal arterial microaneurysm (RAM) rupture or age-related macular degeneration (AMD). Methods: We retrospectively evaluated 47 eyes of 47 patients with SMH due to RAM rupture or AMD who underwent PPV. The presence or development of MHs was confirmed intraoperatively by the surgeon. We compared the incidence of MH between the RAM and AMD groups

This understanding is also lacking in the later segment below

“The MH was closed by initial surgical treatment in all eight cases. In the RAM group, subretinal tissue plasminogen activator (t-PA) injection was performed in 12 eyes. Of the 12 eyes, two developed an MH before t-PA subretinal injection. Of the remaining 10 eyes, four (40%) developed an MH intraoperatively or postoperatively”

First, please make it clear that the MH was closed during the same procedure to remove the vitreous hemorrhage. Initial surgical treatment gives the impression that a planned MH surgery was done.

The sentence “The MH was closed by initial surgical treatment in all eight cases.” was changed to “The MH was closed by same surgical treatment that identified MH in all eight cases.” in response to the reviewer's comment (page8, line152). Abstract was also changed.

Secondly, those eyes that developed MH after t-PA injection should be excluded for the simple reason that the intervention could be the reason for MH and not entirely the disease. So, if the injection was not done, the MH may not have occurred. This point has to be reflected in the manuscript.

Thank you for your suggestion. You have raised an important point; however, we believe that while the incidence of MH due to the disease itself is important, another important message is that subretinal t-PA injection causes MH in RAM, but not in AMD, and therefore requires attention during surgery. We have divided the incidence of MH in the results into those due to the disease itself and those related to the surgical technique in the result (page7 , line121-124 ).We also have added the following text in the discussion to indicate the need for caution with subretinal t-PA injection, particularly in RAM rather than AMD. ” MH has not occurred in patients with AMD who have undergone subretinal t-PA injection in this study. We consider that the risk of MH may be increased when subretinal t-PA infusion is performed in patients with RAM, because patients with SMH due to RAM have a weakened fovea, even if MH did not occur preoperatively. “(page11 , line206-210)

We greatly appreciate both your help and that of the referees concerning improvements to this paper. I hope that the revised manuscript is now suitable for publication.

Sincerely yours,

Riko Matsumoto, MD, corresponding author

Department of Ophthalmology, Shiga University of Medical Science

Seta-Tsukinowacho, Otsu, Shiga 520-2192, JAPAN

Tel: +81-77-548-2276

Fax: +81-77-548-2279

e-mail: rokamoto@belle.shiga-med.ac.jp

Attachment

Submitted filename: Response to Reviewers2.docx

pone.0319266.s004.docx (21.6KB, docx)

Decision Letter 2

Jiro Kogo

30 Jan 2025

Incidence of macular hole in patients undergoing pars plana vitrectomy for submacular hemorrhage

PONE-D-24-29059R2

Dear Dr. Matsumoto

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jiro Kogo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript explores the incidence of MH found during the operation for SMH which is one point that should be considered. Several points have been responded and the revised manuscript has been improved.

Reviewer #3: The authors have addressed the issues raised in previous rounds of review satisfactorily.

This paper can now be accepted for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #3: Yes:  OGUGUA NDUBUISI OKONKWO

**********

Acceptance letter

Jiro Kogo

PONE-D-24-29059R2

PLOS ONE

Dear Dr. Matsumoto,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

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on behalf of

Prof. Jiro Kogo

Academic Editor

PLOS ONE

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