Clinical Observation of Anatomical and Visual Outcomes of Macular Hole after Inverted Internal Limiting Membrane Flap in Patients with Idiopathic Macular

Objective To investigate anatomical and visual outcomes of macular hole (MH) after inverted internal limiting membrane (ILM) flap technique for idiopathic macular hole (IMH). Methods A total of 13 IMH cases diagnosed in Shanxi Eye Hospital between January 2015 and June 2016 were included in the study. All patients underwent vitrectomy combined with indocyanine green-assisted inverted ILM flap technique. The MH closure rate, best-corrected visual acuity (BCVA), changes of ellipsoid zone (EZ), and external limiting membrane (ELM) were examined before operation and one, three, and six months after operation. Furthermore, 488 nm fundus autofluorescence (FAF) and spectral domain optical coherence tomography (SD-OCT) were used to observe the dynamic changes in function of macular area after surgery. Results One month after the surgery, the MH closure rate was 100% and the visual acuity (VA) was stable, with no recurrence. Additionally, the average logMAR BCVA before operation was 1.208 ± 0.158, and this value became 0.877 ± 0.105 one month after the operation, showing a significant decrease. Three months after surgery, the average logMAR BCVA was 0.792 ± 0.103, which was significantly lower than the level one month after the surgery but much higher than that six months after surgery (0.708 ± 0.131). Besides, the diameter of the EZ defect of the postoperative one month, three months, and six months was (1377.46 ± 198.65) μm, (964.62 ± 336.26) μm, and (817.08 ± 442.99) μm, respectively. In postoperative one month, three months, and six months, the diameter of the ELM defect diameter was (969.62 ± 189.92) μm, (649.92 ± 413.15) μm, and (557.62 ± 412.50) μm, respectively. The diameter of both EZ and ELM defects was significantly reduced with the passage of time after surgery. Conclusion Inverted ILM flap technique can reconstruct macular anatomical structure and improve VA. This technique is effective for the treatment of IMH with large MH minimum diameter and base diameter.


Introduction
A macular hole (MH) is a retinal defect that appears in the internal limiting membrane (ILM) and extends to (but not include) the retinal pigment epithelium (RPE). Te prevalence of the disease varies from 0.2% to 0.8% among the public [1]. Idiopathic MH (IMH), accounting for most of MH cases, is a common retinal disease with no clear etiologies and with no association with fundus lesion itself. IMH has a higher prevalence in the elderly and presents as a primary cause of their central visual impairment and visual degeneration [2,3]. Tis disease has long been considered incurable until 1991 when Kelly and Wendel frst applied vitrectomy to successfully cure IMH [4]. Subsequently, in 1997, Eckardt frst introduced internal limiting membrane (ILM) peeling to improve the closure rate of MH [5]. Vitrectomy combined with ILM peeling has qualifed as the standard surgical approach for full-thickness MH, which has achieved a closure rate of 90%-100% and a visual acuity (VA) improvement rate of 85% [6,7]. Te most important factors afecting postoperative IMH closure and visual recovery are the minimum diameters of IMH and base diameter. Patients with the minimum diameter of MH <300 μm and base diameter <1000 μm present a good prognosis [8], but larger values of these two parameters cause a low closure rate and poor prognosis and therefore have formed an obstacle that clinicians have been trying to solve.
In 2010, Michalewska et al. reported the application of inverted ILM fap technique to MHs with a diameter over 500 μm [7] and its satisfactory efcacy in improving VA and closure rate of IMH and myopic MHs [7]. A large-scale comparative study found that the closure rate was 95.6% and 88.4% in patients with IMH >400 μm and myopic MH after the application of the inverted ILM fap technique [9]. Compared with ILM implantation technology, inverted ILM fap technology can better restore the photosensitive layer and obtain better functional results. For larger MHs (>700 μm), flling can not only rebuild the closed environment, but also help the recovery of the hole. However, a meta-analysis showed that in patients with large MHs, vitrectomy with an inverted ILM fap technique had a higher rate of anatomical closure than ILM dissection. Te inverted ILM fap technique had better visual recovery at short-term follow-up, but no advantage was found in longer-term follow-up. For patients with larger MHs, the inverted ILM valve technique should be considered the frst-choice conventional treatment. Terefore, more randomized controlled trials with larger sample sizes and longer follow-up are recommended to confrm the efectiveness of the inverted ILM fap technique.
Optical coherence tomography (OCT) can clearly show retinal microarchitecture and has become the dominant imaging method for the diagnosis of retinopathy as well as the confrmation of the anatomical success after surgery for IMH [10]. Studies have shown that spectral domain OCT (SD-OCT) delineated structural changes in the photoreceptor layer of the eye after surgical closure of IMH, including disruption of the ellipsoidal zone (EZ) and external limiting membrane (ELM) after surgical closure of IMH. However, only a few studies have evaluated the anatomical reconstruction of fovea centralis after using the inverted ILM fap technique in IMH patients and have pointed out recovery from ELM is a key structure feature associated with postoperative bestcorrected VA (BCVA) [11].
We used the inverted ILM fap technique to treat refractory IMH patients with large MHs and then observed anatomical and visual outcomes of MHs, to provide better treatment and sufcient theoretical basis for the treatment of IMH.

Research Subjects.
Inpatients who were diagnosed with IMH in Shanxi Eye Hospital between January 2015 and June 2016 were collected. Patients with minimal hole diameter >500 μm and base diameter >1000 μm were selected and included. However, patients with the following conditions were excluded: (1) patients with other ocular diseases such as myopia, glaucoma, age-related macular degeneration, and uveitis; (2) patients with a history of ocular surgeries; (3) patients without continuous follow-up data. General data of the included patients were recorded. Informed consent was obtained from all patients, and the study had gained the approval of the Ethics Committee of Shanxi Eye Hospital (SXYYLL20211028).

Operation Methods.
All patients in this study were treated by the same surgeon. First, a routine 25-G vitrectomy was performed. Te posterior vitreous membrane was peeled of after intravitreal injection of a small amount of triamcinolone acetonide. Ten, ILM was treated with indocyanine green dye (0.05 mg/mL), and ILM around MH with a diameter of about two optic discs was removed. Superior ILM attached to the margins of MH was left in place, and subsequently, the remnant ILM was inverted upside-down to cover the MH. After air/liquid exchange, the eyes were flled with 14% of C 3 F 8 gas. All patients underwent ultrasonic emulsifcation combined with posterior chamber intraocular lens implantation. After surgery, patients were kept in a face-down position for two weeks.

Evaluation Indexes.
Before the operation and one, three, and six months after the operation, BCVA was detected, and all patients received a slit-lamp examination, SD-OCT (Spectralis OCT of Heidelberg Engineering, Germany), and 488 nm fundus autofuorescence (FAF) examination (HRA-II, Heidelberg Engineering, Germany). SD-OCT was used to scan and measure the minimum diameter of MH and the maximum base diameter before surgery, as well as the defect diameter of EZ and ELM.

Statistical
Analysis. SPSS19.0 statistical software was adopted for statistical analysis in this study. Te measurement data were expressed as mean ± standard deviation (Mean ± SD), and a t-test was used to compare the results from the two groups. P < 0.05 was taken as a statistically signifcant diference.

Inverted ILM Flap Technique Signifcantly Improves
Macular Hole in Patients with IMH. Te average age of the patients was 65.769 ± 3.479. Te patients were examined by SD-OCT and FAF before surgery and one, three, and six months after surgery. Before surgery, the inverted ILM fap and its underlying liquid cavity were visible in the MH, and a high intensity of the FAF signal was also observed (Figures 1(a) and 1(b)). One month after the surgery, the liquid cavity disappeared in the MH, while protuberances demonstrating a moderate signal and particles showing a high signal appeared. Besides, a new area with a low FAF signal was observed (Figures 1(c) and 1(d)). Tree months after surgery, the moderate signal protuberances became larger and new low FAF signal areas were observed between high fuorescence signals (Figures 1(e) and 1(f )). Six months after surgery, the abovementioned protuberances disappeared in the MH, and the ELM, EZ, and RPE layer defects were shown; additionally, the area with a low FAF signal was enlarged (Figures 1(g) and 1(h)).

Inverted ILM Flap Technique Signifcantly Enhanced the
Visual Acuity of IMH Patients. Te postoperative BCVA and MH closure rate was calculated and analyzed in accordance with SD-OCT and FAF examination in a bid to evaluate the therapeutic efect of the inverted ILM fap technique. Te results showed that the minimum diameter of MH before surgery was 680 μm, base diameter 1072 μm, and the logMAR BCVA 1.208 ± 0.158. While one month after the operation, MH closure rate was 100% accompanied with "U"-shaped or "V"-shaped healing, and the condition remained stable during the follow-up period without recurrence; the logMAR BCVA levels of patients were signifcantly reduced, with an average logMAR BCVA of 0.877 ± 0.105; defect diameter of EZ and ELM stood at (1377.46 ± 198.65) μm and (969.62 ± 189.92) μm, respectively. Tree months after the operation, the average logMAR BCVA was 0.792 ± 0.103, the diameter of EZ and ELM defect was (964.62 ± 336.26) μm and (649.92 ± 413.15) μm, respectively; signifcant diferences were found in such indexes between one month and three months after surgery. Six months after the operation, the average logMAR BCVA of all patients was 0.708 ± 0.131, and the diameter of EZ and ELM defect was (817.08 ± 442.99) μm and (557.62 ± 412.50) μm, respectively; signifcant diferences were found in such indexes between three months and six months after surgery. In addition, FAF examination showed that the patient's preoperative fundus fuorescence was strong, while it turned into low signal six months after surgery (Figure 2), suggesting that the inverted ILM fap technique was efective in the treatment of IMH patients.

Discussion
MH <400 μm can be categorized into grades I and II, and its closure rate climbs from 58% to more than 90% after applying diferent treatment techniques combined with vitrectomy [11,12]. However, 44% of patients with MH >400 μm still sufered from MHs opened after conventional vitrectomy combined with ILM peeling [13], thus frequently necessitating a second operation [7]. Refractory MHs usually include MHs >400 μm, pathological myopia combined with macular splitting, MHs lasting more than six months and undergoing multiple surgeries, or MHs with extensive retinal detachment or proliferative vitreoretinopathy. IMH patients included in this study were scanned by SD-OCT and measured to have a minimum diameter >500 μm and a base diameter >1000 μm, which are clinically refractory MHs. Tese patients received vitrectomy combined with inverted ILM fap technique and 100% closure rate. Such outcomes may be attributable to the fact that peeling of part of ILM can relieve the tangential traction of ILM at the MH. In addition, the inverted ILM fap covering the MH allows the hole to form a closed cavity and thus promotes the absorption of fuid in the MH through the RPE cell pump function. Te inverted ILM fap covering the MH also stimulates the proliferation of glial cells in the retina and on the surface of ILM and acts as a scafold for cell proliferation [7,14]. Collectively, the inverted ILM fap technique promotes the closure of MHs.
Intraoperative peeling of the ILM is benefcial in removing completely the contractile tissue attached to it, reducing the tangential traction around the hole, removing the scafold for the proliferation of fbroblasts and RPE cells, preventing the generation of epiretinal membrane, and preventing recurrence. Minor damage during ILM peeling can stimulate Müller cell proliferation and promote hole healing. However, in patients with larger hole diameters, unhealed after ILM peeling, and high myopic macular holes, the hole closure after surgery is higher than low-discovery ILM fap surgery and can promote the healing of postoperative MH healing, increase the closure rate of postoperative hiatus with a diameter >400 μm, and improve patient visual acuity. After ILM fap operation, the ILM in the hole will not act as a foreign body to hinder the closing process of the hole. On the contrary, it will provide a scafold for glial cells and promote the proliferation of glial cells, thereby pulling the photoreceptor cells to move to the center of the hole, promoting the hole is closed. Terefore, for patients who face difculty in closing the hole after routine vitrectomy combined with ILM peeling, it can be combined with ILM fap surgery at the same time during the operation.
FAF can reveal the content and distribution of lipofuscin in RPE cells in a noninvasive manner, refecting the functional status of RPE and photoreceptor cells. In this study, it was shown that the patient presented low FAF signals after surgery. Nonetheless, according to FAF examination results one month after the surgery, it was found that the MH was not closed and ILM was still present, with low signal spots in the macular area in the afected eye; three months after the surgery, OCT showed localized protuberance with moderate signal intensity in the inner retina and particles with high signal intensity in the outer retina, while FAF demonstrated an expansion of the area with low signal intensity. Such imaging outcomes may be the result of glial cell proliferation along the ILM fap [15,16]. Six months after the surgery, the abovementioned protuberance and the high signal particles decreased; additionally, we observed the defect of EZ and RPE layers. Te possible reason for the dynamic changes may be that the inverted ILM fap technique stimulates the activation and proliferation of glial cells such as Müller cells along the scafold provided by the fap, which assisted the repairing and reconstruction of the retina; this inverted ILM fap technique-caused process is found the most obvious from the third to the fourth month [17]. However, while Müller cells are activated and proliferated, retinal microglia may also be activated to exert phagocytic function and produce a large amount of infammatory substances (such as TNF-a, IL-1β) and to exert toxic efects on retinal cells. Consequently, such activation can result in the inhibition of excessive proliferation of glial cells and damage RPE cells [18,19].

Journal of Healthcare Engineering
Te diameter of IMH constitutes an important factor afecting the closure rate and postoperative VA [20]. Currently, OCT is used clinically to measure diferent parameters of MH to predict the closure of MHs and recovery of VA, such as the minimum diameter of the hole, the base diameters, the height of the hole, the angle between the margin of the hole and the base, and the various formulas deduced hereinafter [21,22]. As reported, the minimum diameter and base diameters were important factors affecting postoperative MH closure and visual prognosis [8].
On top of it, the two parameters are easy to measure and calculate. Te visual prognosis was related to "U"-shaped MH healing, normal thickness of fovea centralis, and integrity of photoreceptor layer [23]. In this study, it was found that the defect diameter of both EZ and ELM after surgery had a tendency to decrease during follow-up, indicating the

Conclusion
Inverted ILM fap technique can increase the postoperative closure rate of IMH with a large diameter. Additionally, it acts to improve postoperative VA as well as reconstruct the retinal structure. Terefore, this technique can serve as an efective treatment for MH with large minimum diameter and the large base diameter. However, whether the indocyanine green-assisted inverted ILM fap technique leads to retinal cell damage still requires more observation and research studies.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.  Figure 2: Efect of the inverted internal limiting membrane fap technique on visual acuity in patients with IMH ocular SD-OCTscanning of one patient before surgery (a), one month after surgery (b), three months after surgery (c), six months after surgery (d), ocular FAF images of one patient before surgery (e), and six months after the surgery (f ). Postoperative BCVA and MH closure rate before and after operation (g-i). Yellow arrows indicate protuberance with moderate signal intensity; red arrows denote the range of fuorescence intensity. * P < 0.05, * * P < 0.01, and * * * P < 0.001. Journal of Healthcare Engineering 5