Choroidal and retinal displacements after vitrectomy with internal limiting membrane peeling in eyes with idiopathic macular hole

It has been reported that the macular region of the retina is displaced after vitrectomy with internal limiting membrane (ILM) peeling in eyes with macular hole (MH), but the displacements of the deeper layers of the eye, e.g. RPE and choroid are unclear following the surgery. We used optical coherence tomography (OCT) and OCT angiography (OCTA) to obtain 3 mm × 3 mm en face images before, and 2, 4, and 8 weeks following the vitrectomy with internal limiting membrane (ILM) peeling from 22 eyes of 22 patients with a MH. The OCT and OCTA images showed displacements of the fovea and choroidal intermediate vessels postoperatively. The degree of displacement of the choroid was significantly less than that of the retina (P < 0.001). The displacements of the choroidal bifurcations were significantly correlated to their preoperative distance from the optic disc (r = −0.467, P < 0.001) and they were significantly correlated with the retinal displacements (r = 0.535, P < 0.001). The retina was displaced inferiorly and centripetally, but these localized displacements were not observed in the choroid. In conclusion, clinicians need to be aware of these displacements when evaluating the subfoveal choroid following the surgery because the displacement is different between the retina and the choroid.

Germany) to obtain the 3 × 3 mm 2 en face images of the microvasculature in the macular region 10 . The images of the SCP that was provided automatically by the embedded software of the OCTA were used to measure the displacements of the retina. The images of the ICP obtained from the same area of the 3 × 3 mm 2 en face images using OCT function were used to measure the displacements of the choroid (Fig. 1). The SCP is located between the ILM and the inner plexiform layer, and the ICP is located between 89 and 115 µm posterior to the RPE layer. The two graders (KG and TI) reviewed all of the images, and excluded low quality OCT angiographic images from the analysis. The vascular displacements were measured independently by these two observers who were masked to the other clinical findings.
Measurements of displacement using octA images. The displacements of retina were measured using the OCTA and OCT images as described in detail 10 . In brief, we first selected an easily identifiable retinal vessel bifurcation in the SCP image and choroidal vessel bifurcation in the ICP image. The distance between the bifurcation and the center of the optic disc was measured (#1 in Fig. 2A). Then, the angle formed by a line between the center of the optic disc and the bifurcation point and a horizontal line through the center of the optic disc was measured (#2 in Fig. 2). In addition, the distance between the bifurcation and the fovea was measured (#3 in Fig. 2).

Surgical techniques.
We performed a standard three-port PPV with the Alcon Constellation 25-gauge (G) system (Alcon Laboratories, Inc., Fort Worth, TX, USA). Core vitrectomy was performed, and if necessary, a posterior vitreous detachment was created. Then, the ILM was peeled circumferentially from the retina using ILM-peeling forceps in all cases. Then, we performed a peripheral vitrectomy with shaving and inspected carefully the periphery over 360 degrees carefully in all cases. Finally, fluid-air exchange was performed followed by the injection of 20% sulfur hexafluoride (SF 6 ) or 12% perfluoropropane (C 3 F 8 ) into the vitreous cavity. The patients were instructed to maintain a prone position until a closure of the MH was confirmed with OCT following the surgery.
We performed cataract surgery and implanted a foldable acrylic IOL into the capsular bag on all 17 phakic eyes.
Statistical analyses. We used one-way analysis of variance (ANOVA) to evaluate the significance of the relationship between the displacements in the four quadrants and the chronological data. We used Pearson's correlation coefficient test to evaluate the significance of the associations between the changes in the displacement of each bifurcation and the angle formed and the preoperative distance and angle. All statistical analyses were performed using the Statistical Package for Social Sciences for Windows 21.0 (SPSS Inc, Chicago, IL).

Results
Demographics of patient. Thirty-eight eyes of 38 patients with a MH underwent the successful MH closure surgery during the period. Sixteen eyes were excluded; 2 for prior vitrectomy, 1 for macular degeneration, 10 for high myopia, and 3 for severe cataract that prevented an acquisition of high quality OCTA images. Finally, 22 eyes of 22 patients (mean age, 66.0 ± 9.3 years) were studied. The demographics of the patients and the surgical procedures are shown in Table 1. The mean size of the ILM peeled area was 3.73 ± 0.55 disc diameters (DD). The mean number of retinal bifurcations that was identified was 50.1 ± 8.3 per eye and the mean number of choroidal bifurcations was 19.8 ± 2.5 per eye.   www.nature.com/scientificreports www.nature.com/scientificreports/ changes of foveal position. The fovea was displaced following the surgery, and the changes in the average distance between the fovea and the optic disc were shortened by 0.14 ± 0.06 mm and the angle was rotated downwardly by an average of 0.89 ± 0.73°. changes in distance between retinal or choroidal bifurcations and optic disc. The mean preoperative distance between the retinal bifurcation and the center of the optic disc was 4.46 mm, and it was significantly reduced to 4.32 mm (P < 0.001) at 2 weeks postoperatively. The mean distances were also reduced significantly in all four quadrants, and the reduction was maintained throughout the postoperative period ( Table 2, Fig. 3A).
The mean preoperative distance between the choroidal bifurcations and the center of the optic disc was 4.40 mm, and it was significantly reduced to 4.30 mm (P < 0.001) at 2 weeks postoperatively. The mean distances were also reduced significantly in all four quadrants, and the reductions were maintained throughout the postoperative period. No significant differences were observed in the distances during postoperative periods in the both of retina and choroid in all of the quadrants (Fig. 3B). A significant difference was observed in the distance in the both of retina and choroid among the 4 quadrants at Week 2 (P < 0.001; Fig. 3B). The displacement of each choroidal bifurcation at Week 2 was significantly correlated with its preoperative distance from the choroidal bifurcation to the optic disc (r = −0.467, P < 0.001; Fig. 4A). changes in angle of retinal and choroidal vessel bifurcations. The mean preoperative angle formed by a line between the center of the optic disc and the retinal bifurcation point and a horizontal line through the center of the optic disc was −7.9°, and it was significantly rotated downwardly to −8.6° at 2 weeks following surgery (P < 0.001, Fig. 3C). In addition, the angle was significantly rotated downward in the temporal, superior, and nasal quadrants at 2 weeks following surgery (P < 0.01). A significant difference was observed in the angle among the 4 quadrants at Week 2 following surgery (P < 0.001; Fig. 3D). The changes in the angles were not altered significantly during the postoperative period.
On the other hand, the mean preoperative angle formed by a line between the center of the optic disc and the choroidal bifurcation point and a horizontal line through the center of the optic disc was −7.9°, and it was changed to −8.1° at Week 2 which was not a significant. The changes in the angle at each choroidal bifurcation at Week 2 were not significantly correlated with the preoperative angle to the optic disc (Fig. 4C). changes in distance between retinal and choroidal bifurcations and fovea. The changes in the distance between the fovea and the retinal vessel bifurcations was significantly smaller in the temporal, superior, and inferior quadrant following the surgery (Fig. 3E, Table 2). On the other hand, the distance between the fovea and the choroidal vessel bifurcations was not significantly changed in all of the quadrants following the surgery. However, the distance between the fovea and the bifurcations of the choroidal vessel was in opposite directions between the superior, temporal quadrants and nasal, inferior quadrants (Fig. 3F).

Quadrant
Week 2  Table 2. Changes in distance and angle between bifurcations and the optic disc or the fovea.
OCT and FAF findings following surgery. The swept source-OCT images following the surgery with ILM peeling showed a successful closure of the MH and the displacement of fovea and choroidal intermediate vessels (Fig. 5). Hyperfluorescent lines were not observed in the FAF image in any of the eyes following the surgery (Fig. 6).

Discussion
Our results showed that the retina was displaced nasally, downwardly, and centripetally toward the center of the optic disc in the macular region. These findings corroborate the findings of an earlier report 10 . The OCT images of the intermediate choroidal slab showed that the choroid was also displaced nasally following the surgery in eyes with an IMH. The distance between the choroidal bifurcations and the optic disc was significantly decreased in all four quadrants following the surgery. In addition, these distances were positively and significantly correlated with the corresponding preoperative distance between the optic disc and the choroidal bifurcations. These findings indicate that the bifurcations farther from the optic disc had greater displacements following the surgery. In addition, the analyses of the OCT horizontal B scan images showed that the intermediate choroidal vessels were   www.nature.com/scientificreports www.nature.com/scientificreports/ displaced toward the optic disc following the surgery although the degree of the displacement was less than that in the retina.
On the other hand, the angle formed by a line between the center of the optic disc and the retinal or choroidal bifurcation and a horizontal line through the center of the optic disc was not changed following the surgery. In addition, the change in the angle formed by the line from the choroidal bifurcation at week 2 was not significantly correlated with the preoperative angle and was not significantly different in all of the four quadrants. Those results indicate that the choroid was not significantly displaced downwardly following the surgery which differs from the significant downward displacement in the retina.
In the displacement measurements of the choroid using the fovea as the fixed point, the distance between the bifurcations of the choroidal vessel and the fovea was not changed following the surgery. In addition, the amount of displacement of the choroidal bifurcations was not correlated with the preoperative distance between the choroidal bifurcations and the fovea. These results suggest that the choroid was not displaced centripetally following the surgery which differs from the significant displacement of the retina.
Taken together, the choroid was significantly displaced toward the optic disc following the vitrectomy with ILM peeling in eyes with an IMH, but not downwardly, and centripetally. The distance of displacement of the choroid was significantly correlated with that of the retina in each quadrant which suggests that the displacement of the choroid toward the optic disc was corelated to the displacement of the retina.
There have been studies on the retinal displacements in the macular region in eyes following MH closure using ILM peeling with fundus photographs 7-9 , or OCTA en face images 10 . The retinal vasculature is mainly located in the inner retina, meaning that the macular displacement indicates an inner retinal displacement.
There are several reasons why the inner retina is displaced following vitrectomy with ILM peeling. The ILM is the basal membrane of the Müller cells, and it contributes to the integrity of the retina 21 and less to its elasticity 11,22 . The ILM has about 1,000-fold greater strength mechanically than the other cellular layers, and the ILM comprises 50% of the retinal rigidity 21,23 . In addition, the optic nerve fibers are connected to the lamina cribrosa 24 , and the retinal nerve fibers could move toward the optic disc following a contraction of the nerve fibers. Therefore, the removal of the ILM causes a lack of the structural support of the retina which probably causes the contraction of the nerve fibers where the ILM was peeled and the displacement in the inner retina toward the optic disc.
We were not able to find any studies reporting on the presence of hyperfluorescent lines following the surgery. Shiragami et al. reported that hyperfluorescent lines were observed superiorly and in parallel with the retinal vessels in 27 of the 43 eyes (62.8%) following surgery in eyes with a rhegmatogenous retinal detachment (RRD). dell'Omo et al. reported that displacements of retina were observed in 44 of 125 (35.2%) eyes following a retinal reattachment in eyes with a RRD 25 . These findings indicated that hyperfluorescent lines can be observed after the inner retinal layer is displaced laterally from the RPE layer as in eyes with a RRD. Our results suggest that the inner retinal layer was not displaced from the RPE layer, and the RPE layer was displaced together with the inner retinal layer following the surgery.
The Müller cells are believed to maintain the structural integrity of the retina as an important role 26 . The sensory retina adheres strongly to the RPE which allows the RPE to transport subretinal fluid at a very high rate against a substantial gradient of hydrostatic or osmotic pressure 27 . This would be related to the observation that the RPE layer was displaced along with the inner retinal layer.
The choroid is comprised mostly of blood vessels histologically and is divided in five layers; Bruch's membrane, the choriocapillaris, Haller's layer, Sattler's layer, and the suprachoroid 28 . The choriocapillaris is approximately 10 µm thickness in the foveal region, and it is a highly anastomosed network of capillaries 28 . The choriocapillaris can be detected by OCTA, but it is difficult to evaluate the displacements of the choriocapillaris layer because its hexagonal and lobular shapes resembles that of the RPE. However, the choriocapillaris layer is presumably not displaced from the RPE layer because the choriocapillaris layer is apposed to the outermost layer of Bruch's membrane which adheres to the RPE.
OCT en face image can detect the vasculature in the choroidal vessels of Haller's and Sattler's layer. However, the choroid has the stroma containing extravascular tissue, e.g. elastic fibers and collagen. This means that the choroidal vessels are not adhesively connected in these layers and to the choriocapillaris layer which might cause the gap in the displacement of the choriocapillaris layer and the choroidal vessels in the OCT images. The gap might lead to the differences in the displacements between the retina and choroid following surgery.
Taken together, the peeling of the ILM probably causes the displacement of the inner retina and the degree of displacement would be related to the displacement of the RPE and choroid. However, there is no adhesive connection between the intermediate choroidal vessels causing a gap in the displacement between the choroid and the inner retina following the surgery.
Our result showed that the choroid was not displaced centripetally, i.e., toward the fovea, following the MH closure which was reasonable because if the choroid and the RPE were displaced toward the fovea, then some wrinkling should be observed in the RPE layer over the fovea following the surgery. The distance between the fovea and the bifurcations of the choroidal vessel was in opposite directions between the superior, temporal quadrants and nasal, inferior quadrants. These differences should be related to the fact that the fovea was displaced nasally and inferiorly following the surgery. The displacement of the choroid was less than that of the retina which can cause the distance to be shorter between the fovea and the nasal and inferior bifurcations of the choroid.
There have been many reports about the choroidal thickness and blood flow at the fovea. However, the choroid is displaced following the surgery, and the degree of the displacement in choroid is different from that in retina. This means that the subfoveal choroid following surgery is different from that before surgery, and it can cause differences of the measured area of the subfoveal choroid between before and following surgery. Accordingly, we need to pay attention to the results of the subfoveal choroid, e.g. thickness and blood flow following the vitrectomy with ILM peeling in any kind of retinochoroidal diseases.
This study has several limitations. First, this was a retrospective study on a limited number of participants, which might be the reason why no significant differences were found in the angle and the distance to the fovea in the choroidal displacement. Second, the displacement of choroid was compared with that of retina in each quadrant, and because it is rare that the location of retinal bifurcation is coincident with that of choroidal bifurcation, we did not compare the displacement of retina and choroid directly for each bifurcation. Third, although we confirmed the choroidal displacement in the OCT B-scan images, and we did not measure the distance of displacement using the images because it is difficult to find a fixed point on the image to measure. Fourth, analyses of the 6 × 6 mm 2 en face OCTA image would enable us to analyza a wider field of view. However, the resolution is better with the 3 × 3 mm 2 en face images. Thus, we used the 3 × 3 mm 2 en face images to determine the displacements. Further prospective studies on analyses of the different choroidal vascular layers using a larger number of subjects will be necessary for clarification of the mechanism of the choroidal displacements.
In conclusion, the choroid was displaced toward the optic disc following the vitrectomy with ILM peeling in eyes with a MH. Clinicians need to be aware of this when evaluating the subfoveal choroid following the surgery because the displacement is different between the retina and the choroid.