Purpose
To compare immunocytochemical and ultrastructural characteristics of “lamellar hole–associated epiretinal proliferation” in lamellar macular holes with “conventional epiretinal membrane” in macular pseudoholes.
Design
A consecutive observational case series, laboratory investigation.
Methods
We analyzed surgically excised flat-mounted internal limiting membrane specimens and epiretinal membrane specimens removed from 25 eyes of 25 patients with lamellar macular holes (11 eyes) and macular pseudoholes (14 eyes) using interference and phase-contrast microscopy, immunocytochemistry, and transmission electron microscopy. By spectral-domain optical coherence tomography, epiretinal material of homogenous reflectivity without contractive properties was categorized as lamellar hole–associated epiretinal proliferation, whereas tractional epiretinal membranes presenting contractive properties were termed conventional epiretinal membrane.
Results
Lamellar hole–associated epiretinal proliferation was seen in 73% of eyes with lamellar macular hole. Eyes with macular pseudohole presented with conventional epiretinal membrane. In lamellar hole–associated epiretinal proliferation, positive immunoreactivity for anti–glial fibrillary acidic protein, hyalocyte markers, and anti–collagen type I and III was seen. In contrast, specimens of macular pseudoholes were positive for α-smooth muscle actin and anti–glial fibrillary acidic protein, predominantly. Cellular ultrastructure showed that lamellar hole–associated epiretinal proliferation of lamellar macular holes mainly consisted of fibroblasts and hyalocytes, whereas myofibroblasts dominated in conventional epiretinal membranes of macular pseudoholes.
Conclusions
Cells within lamellar hole–associated epiretinal proliferation appear to originate from vitreous and possess less contractive properties than cells of conventional epiretinal membranes. Our findings point to differences in pathogenesis in a subgroup of lamellar macular holes presenting lamellar hole–associated epiretinal proliferation on the retinal surface.
Recently, high-resolution optical coherence tomography (OCT) studies have not only shown epiretinal membranes in lamellar macular holes but also epiretinal proliferation with unusual appearance. First described by Witkin and associates, unusual epiretinal proliferation in lamellar macular holes was demonstrated as a highly reflective line with moderately reflective material filling the space between the inner border of the epiretinal membrane and the retinal nerve fiber layer on OCT images. Parolini and associates also reported on unusual epiretinal proliferation in eyes with lamellar macular hole in a clinicopathologic case series, and named it “dense” membranes that differ from “tractional” membranes according to their morphologic features. Since there is no widely accepted terminology, Pang and associates introduced the term “lamellar hole–associated epiretinal proliferation” to characterize the thick homogenous layer of material with medium reflectivity on the epiretinal surface in eyes with lamellar macular holes. Of note, the presence of lamellar hole–associated epiretinal proliferation was recently shown to be related to the presence of photoreceptor layer defects and poor visual acuity.
According to our current knowledge of the pathogenesis of vitreomaculopathies, any epiretinal tissue might have the potential to exert traction onto inner retinal layers. However, lamellar hole–associated epiretinal proliferation does not appear to have contractive properties, in contrast to conventional epiretinal membranes. In eyes with macular pseudoholes, generation of tractional forces by conventional epiretinal membranes becomes visible as retinal folds. Consequently, lamellar macular holes and macular pseudoholes appear to have a different pathogenesis, which in turn might explain the different clinical course of these 2 entities. Eyes with lamellar macular hole have mostly been seen as stable conditions over a long period of time, and were shown not to respond to surgery as well as eyes with macular pseudohole.
Furthermore, lamellar hole–associated epiretinal proliferation was recently suggested to be primarily driven by a proliferation of Müller cells onto the inner retina, originating from the middle layers of the retina. This hypothesis is in accordance with histopathologic findings of Parolini and associates, who presented cells of positive immunoreactivitiy for glial fibrillary acidic protein in lamellar hole–associated epiretinal proliferation. But there is less detail on the cell and collagen composition and topography of lamellar hole–associated epiretinal proliferation compared to conventional epiretinal membranes in macular pseudoholes. Therefore, the aim of this study was to analyze immunocytochemical and ultrastructural characteristics of lamellar hole–associated epiretinal proliferation in eyes with lamellar macular hole, and to compare with conventional epiretinal membranes in eyes with macular pseudohole by using fluorescence microscopy and transmission electron microscopy.
Methods
This is an interventional clinicopathologic case series of surgically excised epiretinal membrane and internal limiting membrane specimens removed from 25 eyes of 25 patients with lamellar macular hole (11 eyes) and macular pseudohole (14 eyes) who underwent vitrectomy at the Ludwig-Maximilians-University, Department of Ophthalmology, between January 2011 and March 2013. All specimens were consecutively harvested by 2 surgeons and prepared for flat-mount preparation, immunocytochemistry, phase contrast microscopy, and interference microscopy, as well as transmission electron microscopy. Patients with insufficiently good quality of OCT images were excluded, as were those without follow-up for a minimum of 3 months. For clinical analysis patients’ records were reviewed for age, sex, preoperative best-corrected visual acuity (BCVA), postoperative BCVA, period of follow-up, pre- and postoperative state of the lens, and the presence of metamorphopsia. The Institutional Review Board and the Ethics Committee of the Ludwig-Maximilians-University Munich approved the retrospective review of the patients’ data, as well as the histopathologic preparation and analysis of the patients’ specimens (No 471-14). Informed consent was obtained from each patient. The study was conducted according to the tenets of the Declaration of Helsinki.
Spectral-Domain Optical Coherence Tomography Examination
For spectral-domain optical coherence tomography (Spectralis OCT; Heidelberg Engineering, Heidelberg, Germany) analysis, we retrospectively reviewed and reevaluated each volume B-scan of baseline visits. According to their appearance and reflectivity, epiretinal membranes were classified by spectral-domain optical coherence tomography as previously published. Epiretinal membranes with contractive properties were termed “conventional epiretinal membrane.” In spectral-domain optical coherence tomography, they appear as a thin hyperreflective layer exerting traction on the inner retinal layers, represented as visible retinal folds. In accordance with Pang and associates, epiretinal material of homogenous medium reflectivity without any contractive properties on the retinal surface was termed “lamellar hole–associated epiretinal proliferation.”
The presence of vitreomacular adhesion and the state of posterior vitreous detachment was analyzed by reviewing all OCT scans from each examination.
Surgical Procedure
Patients were recommended to surgery according to the following indication criteria: (1) BCVA decreased to logMAR 0.3 or more, (2) BCVA decreased 2 Snellen lines or more during the preoperative follow-up period, and (3) the patient experienced a significant impairment of the quality of life or a subjective increase of metamorphopsia.
All patients underwent a standard 23 gauge pars plana vitrectomy with sequential internal limiting membrane and epiretinal membrane peeling. If necessary, a posterior vitreous detachment was induced by suction with the vitrectomy probe around the optic nerve head. The posterior hyaloid was detached from the retina, and posterior vitreous detachment was extended to the periphery. Epiretinal membranes and internal limiting membranes were sequentially peeled using an end-gripping forceps. For internal limiting membrane peeling, a vital dye of 0.25 mg/mL solution of Brilliant Blue (Brilliant Peel; Fluoron GmbH, Neu-Ulm, Germany) was used. Removal of the internal limiting membrane was intended to be an area of at least 1 disc diameter surrounding the lamellar macular hole or macular pseudohole. Conventional epiretinal membranes presented as rigid membranes that were easy to grasp during vitrectomy, whereas lamellar hole–associated epiretinal proliferation mostly consisted of yellow, dense tissue with fluffy consistency that was not easy to grasp. The vitreous cavity was filled with a tamponade of either 15% hexafluoroethane (C 2 F 6 ) gas-air mixture, air, or balanced salt solution. Individually, patients were instructed to keep a face-down position for 2 days.
Immunocytochemistry
On average, 2 specimens (range, 1–4) were harvested from each patient. There was no selection of specimens before the preparation procedure. If more than 2 specimens were removed from 1 eye, all specimens were prepared and analyzed. If fewer than 3 specimens were removed, large specimens were segmented for labeling combinations of all 9 primary antibodies.
Immediately after harvesting, the specimens were placed into a 2% paraformaldehyde solution for fixation. For flat-mount preparation, fixated specimens were flattened and unfolded onto glass slides to show the maximum area of their surface using a stereomicroscope (MS 5; Leica, Wetzlar, Germany). Antifading mounting medium 4′,6-diamidino-2-phenylindole (DAPI; AKS-38448; Dianova, Hamburg, Germany) was used to stain cell nuclei, and a cover slide was added.
Interference and phase contrast microscopy was performed with a modified fluorescence microscope (Leica DM 2500, Wetzlar, Germany) at magnifications between ×50 and ×400. For photographic documentation a digital camera was used (ProgRes CF; Jenoptik, Jena, Germany).
Performing immunohistochemistry, primary antibodies were used for glial and retinal cells (anti–glial fibrillary acidic protein [anti-GFAP] and anti-vimentin; DAKO, Hamburg, Germany), for myofibroblasts (anti-α-smooth muscle actin [anti-α-SMA]; Santa Cruz Biotechnology, Heidelberg, Germany), for hyalocytes (anti-CD45 and anti-CD64; Santa Cruz Biotechnology, Heidelberg, Germany), for basal membrane ILM (anti-laminin; DAKO, Hamburg, Germany), and for extracellular matrix (anti–collagen type I; Santa Cruz Biotechnology, Heidelberg, Germany; anti–collagen type II and anti–collagen type III; Biotrend, Cologne, Germany). Manufacturer’s instructions were followed exactly. The specimens were labeled with combinations of 3 primary antibodies. As secondary antibody we used either donkey anti-rabbit Cy2, donkey anti-mouse Cy3, or donkey anti-mouse Cy5 (Dianova, Hamburg, Germany).
Indirect immunocytochemistry comprised the following steps: rinsing in 0.1 M phosphate-buffered saline (PBS, pH 7.4); incubation with 0.1% pepsin in 0.1 M PBS (room temperature, 10 minutes); rinsing twice in 0.1 M PBS (pH 7.4); incubation in normal donkey serum (1:20) in 0.1M PBS, 0.5% BSA, 0.1% Triton X-100, and 0.1% Na-azide (PBTA) (3 hours); incubation with primary antibody in PBTA (room temperature, overnight); rinsing 3 times in PBS (pH 7.4, 10 minutes each); incubation with secondary antibody (each in 1:100 PBS, room temperature, 2 hours); rinsing 4 times in PBS (10 minutes each); postfixation in 0.2% glutaraldehyde solution in 0.1 M PBS (5 minutes); and rinsing 3 times in PBS (5 minutes each).
Preparing negative controls, the primary antibody was substituted with both diluent and isotype controls (IgG2a monoclonal mouse antibodies, X0934; DAKO, Hamburg, Germany; M5409; Sigma-Aldrich, Taufkirchen, Germany). All other procedures were identical to the procedures illustrated above.
Transmission Electron Microscopy
For ultrastructural analysis, all specimens were prepared for transmission electron microscopy. After postfixation (osmium tetroxide 2%, Dalton’s fixative), dehydration in graded concentrations of ethanol and embedding in Epon 812 was performed. Analyzing morphologic features more detailed in both groups of lamellar macular hole and macular pseudohole, each of 5 specimens were additionally prepared for fixation with phosphate-buffered 4% glutaraldehyde solution. For staining of semi-thin sections of 400 nm an aqueous mixture of 1% toluidine blue and 2% sodium borax was used. Ultrathin series sections of 60 nm followed and were contrasted with uranyl acetate and lead citrate. Using a Zeiss light microscope and a Zeiss EM 9 S-2 electron microscope (Zeiss, Jena, Germany), 5 grids (each with 6–9 ultrathin sections) per specimen have been imaged and evaluated by 2 experienced examiners.
In 5 cases, more than 3 specimens were harvested during vitrectomy. Specimens that were not used for immunocytochemistry were directly placed into 4% glutaraldehyde fixation and prepared for transmission electron microscopy.
Results
Clinical Data Analysis
In this study, we included 25 eyes (11 lamellar macular holes and 14 macular pseudoholes) that underwent vitrectomy with epiretinal membrane removal and internal limiting membrane peeling. Among these there were 12 eyes of women (5 lamellar macular holes and 7 macular pseudoholes) and 13 eyes of men (6 lamellar macular holes and 7 macular pseudoholes). At time of surgery, patients’ mean age was 70 ± 7 years (median 70 years; range, 47–85 years). The mean age of patients with lamellar macular hole was 67 ± 9 years (median 70 years; range, 47–85 years). Considering eyes with macular pseudohole, patients’ mean age was 70 ± 4 years (median 70 years; range, 62–77 years). There was no statistical difference in mean age of eyes with lamellar macular hole and macular pseudoholes. Before vitrectomy, 12 patients (6 lamellar macular holes and 6 macular pseudoholes) complained of metamorphopsia. Table 1 shows the clinical data of patients with lamellar macular hole and macular pseudohole, including sex, age, diagnosis, metamorphopsia, preoperative and postoperative BCVA, preoperative and postoperative state of the lens, and the period of follow-up, in months.
| No | Sex/Age | Diagnosis | Metamorphopsia (+/−) | Preoperative BCVA (logMAR) | Postoperative BCVA (logMAR) | Preoperative State of the Lens | Postoperative State of the Lens | Follow-up (mo) |
|---|---|---|---|---|---|---|---|---|
| 1 | M/74 | LMH | − | 0.5 | 0.1 | Phakic | IOL | 21 |
| 2 | M/71 | LMH | + | 0.3 | 0.4 | Phakic | IOL | 18 |
| 3 | F/63 | LMH | − | 0.4 | 0.2 | Phakic | IOL | 16 |
| 4 | F/74 | LMH | + | 0.7 | 0.5 | IOL | IOL | 22 |
| 5 | M/74 | LMH | + | 0.4 | 0.2 | Phakic | IOL | 11 |
| 6 | M/73 | LMH | + | 0.4 | 0.2 | Phakic | IOL | 3 |
| 7 | F/47 | LMH | − | 0.5 | 0.1 | Phakic | IOL | 11 |
| 8 | F/85 | LMH | − | 0.5 | 0.7 | Phakic | IOL | 3 |
| 9 | M/64 | LMH | + | 0.3 | 0.1 | Phakic | IOL | 7 |
| 10 | M/71 | LMH | − | 0.3 | 0.4 | IOL | IOL | 3 |
| 11 | M/66 | LMH | + | 0.5 | 0.5 | Phakic | IOL | 3 |
| 12 | F/76 | MPH | + | 0.4 | 0,0 | Phakic | IOL | 22 |
| 13 | M/73 | MPH | + | 0.4 | 0,1 | Phakic | IOL | 17 |
| 14 | M/79 | MPH | + | 0.7 | 0,4 | Phakic | IOL | 9 |
| 15 | F/65 | MPH | + | 0.1 | 0.1 | Phakic | Phakic | 10 |
| 16 | F/70 | MPH | − | 0.4 | 0.4 | Phakic | IOL | 4 |
| 17 | M/69 | MPH | − | 0.3 | 0.1 | IOL | IOL | 3 |
| 18 | M76 | MPH | + | 0.2 | 0.2 | IOL | IOL | 9 |
| 19 | M/72 | MPH | − | 0.7 | 0,4 | Phakic | IOL | 3 |
| 20 | F/70 | MPH | − | 0.2 | 0.0 | Phakic | IOL | 6 |
| 21 | M/77 | MPH | − | 0.5 | 0.2 | Phakic | Phakic | 9 |
| 22 | F/69 | MPH | + | 0.2 | 0.0 | Phakic | IOL | 4 |
| 23 | F/64 | MPH | − | 0.2 | 0.2 | Phakic | IOL | 3 |
| 24 | M/77 | MPH | − | 0.4 | 0.4 | Phakic | IOL | 4 |
| 25 | F/72 | MPH | − | 0.2 | 0.2 | Phakic | IOL | 3 |
Before surgery, eyes with lamellar macular hole showed a median BCVA of logMAR 0.40 (mean 0.43 ± 0.12 standard deviation [SD]). After vitrectomy, median BCVA increased to logMAR 0.20 (mean 0.30 ± 0.20 SD) during a mean follow-up period of 10.7 months (median 11 months; range, 3–22 months). The difference was statistically significant (Wilcoxon test, P = .047). Of the 11 patients with lamellar macular hole, 1 was found with unchanged BCVA and 2 decreased in BCVA.
Median BCVA of eyes with macular pseudohole was preoperatively logMAR 0.35 (mean 0.35 ± 0.19 SD) and increased postoperatively to median BCVA of logMAR 0.20 (mean 0.19 ± 0.15 SD). Mean follow-up period was 7.1 months (median 4 months; range, 3–22 months). The difference was also statistically significant (Wilcoxon test, P = .010). In this group, 5 of 14 patients were found with unchanged BCVA, whereas no patient lost vision. Comparing eyes with lamellar macular hole and macular pseudohole, there was no statistical difference in improving of BCVA before vitrectomy and final follow-up (Mann Whitney test, P > .05).
At time of surgery, 4 eyes (2 lamellar macular holes and 2 macular pseudoholes) were pseudophakic. From the remaining 21 eyes, 19 eyes (9 lamellar macular holes and 10 macular pseudoholes) underwent combined vitrectomy with cataract extraction and intraocular lens implantation and 2 eyes with macular pseudohole underwent vitrectomy only. Overall, 2 eyes with macular pseudohole remained phakic at time of last follow-up.
Postoperatively, none of the eyes developed a full-thickness macular hole. Regarding foveal contour, 8 of 11 eyes with lamellar macular hole showed a regular foveal contour at last follow-up, and the remaining 3 eyes with lamellar macular hole showed an irregular foveal contour. In the group of macular pseudoholes, 8 of 14 eyes were seen with a normal foveal contour at last follow-up, whereas 6 eyes showed an irregular foveal contour. There was no persistent macular edema noted.
Spectral-Domain Optical Coherence Tomography Analysis
Table 2 shows spectral-domain OCT analysis of eyes with lamellar macular hole and macular pseudohole. Using high-resolution spectral-domain optical coherence tomography, epiretinal tissue was noted in all eyes with lamellar macular hole. Lamellar hole–associated epiretinal proliferation was present in 27% of eyes with lamellar macular hole (3 of 11 eyes), being located on the edges of the macular defect ( Figure 1 , Top left and Top right). A conventional epiretinal membrane alone was noted in 27% of eyes with lamellar macular hole (3 of 11 eyes), mostly presenting eccentric from the fovea. In 46% of eyes with lamellar macular hole (5 of 11 eyes), a combination of both lamellar hole–associated epiretinal proliferation and conventional epiretinal membrane was seen ( Figure 2 , Top left and Top right). Comparing these 3 groups of eyes with lamellar macular holes, there was no statistical difference in median BCVA (Mann-Whitney test, P > .05).
| No | Diagnosis | SD OCT Analysis | Area of Peeled ILM (mm 2 ) | No of Cells | Cell Density (cells/mm 2 ) | Cell Distribution | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| State of Posterior Hyaloid | ERM | LHEP | Single Cells | Homogenous Layer of Cells | Cluster of Cells | |||||
| 1 | LMH | Complete PVD | x | – | 6.9 | 2246 | 326 | – | x | – |
| 2 | LMH | Complete PVD | x | x | 13.32 | 4942 | 371 | – | x | – |
| 3 | LMH | Complete PVD | x | – | 15.44 | 2651 | 172 | – | x | – |
| 4 | LMH | Complete PVD | x | x | 3.62 | 2583 | 714 | – | x | – |
| 5 | LMH | Partial PVD | – | x | 6.74 | 779 | 116 | – | x | – |
| 6 | LMH | Complete PVD | x | x | 7.06 | 867 | 123 | – | x | – |
| 7 | LMH | Complete PVD | x | – | 1.63 | 405 | 248 | – | x | – |
| 8 | LMH | Partial PVD | – | x | 1.93 | 175 | 91 | – | – | x |
| 9 | LMH | Complete PVD | – | x | 2.27 | 322 | 142 | – | – | x |
| 10 | LMH | Complete PVD | x | x | 1.81 | 995 | 550 | – | x | – |
| 11 | LMH | Complete PVD | x | x | 2.24 | 898 | 401 | – | x | – |
| 12 | MPH | Complete PVD | x | – | 34.86 | 17 771 | 510 | – | x | – |
| 13 | MPH | Complete PVD | x | – | 4.21 | 600 | 143 | – | – | x |
| 14 | MPH | Complete PVD | x | – | 21.13 | 267 | 13 | – | – | x |
| 15 | MPH | Complete PVD | x | – | 13.08 | 2510 | 192 | – | x | – |
| 16 | MPH | Complete PVD | x | – | 2.96 | 2525 | 853 | – | x | – |
| 17 | MPH | Complete PVD | x | – | 3.29 | 1772 | 539 | – | – | x |
| 18 | MPH | Complete PVD | x | – | 3.96 | 607 | 153 | – | x | – |
| 19 | MPH | Complete PVD | x | – | 1.85 | 1535 | 830 | – | x | – |
| 20 | MPH | Complete PVD | x | – | 0.91 | 206 | 226 | – | – | x |
| 21 | MPH | Complete PVD | x | – | 2.95 | 1186 | 402 | – | x | – |
| 22 | MPH | Complete PVD | x | – | 5.2 | 985 | 189 | – | x | – |
| 23 | MPH | Complete PVD | x | – | 15.82 | 3401 | 215 | – | x | – |
| 24 | MPH | Partial PVD | x | – | 4.09 | 889 | 217 | x | – | – |
| 25 | MPH | Complete PVD | x | – | 4.2 | 1669 | 397 | – | x | – |
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