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Lamellar Hole–Associated Epiretinal Proliferation in Comparison to Epiretinal Membranes of Macular Pseudoholes
Accepted Manuscript Lamellar hole - associated epiretinal proliferation in comparison to epiretinal membranes of macular pseudoholes Denise Compera, MD, Enrico Entchev, MD, Christos Haritoglou, MD, Renate Scheler, MTA, Wolfgang J. Mayer, MD, Armin Wolf, MD, Anselm Kampik, MD, Ricarda G. Schumann, MD PII:
S0002-9394(15)00277-9
DOI:
10.1016/j.ajo.2015.05.010
Reference:
AJOPHT 9331
To appear in:
American Journal of Ophthalmology
Received Date: 9 March 2015 Revised Date:
11 May 2015
Accepted Date: 12 May 2015
Please cite this article as: Compera D, Entchev E, Haritoglou C, Scheler R, Mayer WJ, Wolf A, Kampik A, Schumann RG, Lamellar hole - associated epiretinal proliferation in comparison to epiretinal membranes of macular pseudoholes, American Journal of Ophthalmology (2015), doi: 10.1016/ j.ajo.2015.05.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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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 analysed 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.
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Lamellar hole - associated epiretinal proliferation in comparison to epiretinal membranes of macular pseudoholes
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Denise Compera, MD; Enrico Entchev, MD; Christos Haritoglou, MD; Renate Scheler, MTA; Wolfgang J Mayer, MD; Armin Wolf, MD; Anselm Kampik, MD; Ricarda G Schumann, MD Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany
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Short Title: Lamellar hole-associated epiretinal proliferation
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Denise Compera, MD Ludwig-Maximilians-University, Department of Ophthalmology, Mathildenstrasse 8, 80336 Munich, Germany Tel.: +49-89-5160 3811 Fax: +49-89-5160 5160 Email: [email protected]
[1]
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INTRODUCTION 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.1-8 First described by Witkin et al., 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 fibre layer on OCT images.2 Parolini et al. also reported on unusual epiretinal proliferation in eyes with lamellar macular hole in a clinical-pathological case series, and named it “dense” membranes that differ from “tractional” membranes according to their morphologic features.3 Since there is no widely accepted terminology, Pang et al. 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.7-8 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.6-8 According to our current knowledge of the pathogenesis of vitreo-maculopathies, 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.3,6-8 In eyes with macular pseudoholes, generation of tractional forces by conventional epiretinal membranes become 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 two 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.5,6,11 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.7 This hypothesis is in accordance to histopathological findings of Parolini et al. 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 in contrast to conventional epiretinal membranes in macular pseudoholes. Therefore, the aim of this study was to analyse 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 clinical-pathological 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 two surgeons and prepared for flat-mount preparation, [2]
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immunocytochemistry, phase-contrast, 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, gender, preoperative BCVA, postoperative BCVA, period of follow-up, pre- and postoperative state of the lens, as well as 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 histopathological 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.
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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-scans of baseline visits. According to their appearance and reflectivity, epiretinal membranes were classified by Spectral-domain optical coherence tomography as previously published.1-2,9 Epiretinal membranes with contractive properties were termed ‘conventional epiretinal membrane’. In Spectral-domain optical coherence tomography, they appear as a thin hyper-reflective layer exerting traction on the inner retinal layers represented as visible retinal folds. In accordance to Pang et al., epiretinal material of homogenous medium reflectivity without any contractive properties on the retinal surface was termed ‘lamellar hole-associated epiretinal proliferation’.7-8 The presence of vitreomacular adhesion and the state of posterior vitreous detachment was analysed by reviewing all OCT scans from each examination.
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Surgical procedure Patients were recommended to surgery according to the following indication criteria: (1) best-corrected visual acuity (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 detaching 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 endgripping 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 one 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 being not easy to grasp. The vitreous cavity was filled with a tamponade of either 15% hexafluoroethane (C2F6) gasair mixture, or air, or balanced salt solution. Individually, patients were instructed to keep a face-down position for two days. [3]
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Immunocytochemistry On average, two specimens (range 1-4) were harvested from each patient. There was no selection of specimens before the preparation procedure. If more than two specimens were removed from one eye, all specimens were prepared and analysed. If less than three specimens were removed, large specimens were segmented for labelling combinations of all nine 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 x50 and x400. For photo 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 antiCD64, 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 anticollagen type III, Biotrend, Cologne, Germany). Manufacturer`s instructions were followed exactly. The specimens were labelled with combinations of three primary antibodies. As secondary antibody we used either donkey anti-rabbit Cy2, donkey antimouse Cy3, or donkey anti-mouse Cy5 (Dianova, Hamburg, Germany). Indirect immunocytochemistry comprised the following steps: rinsing in 0.1M phosphate-buffered saline (PBS, pH 7.4), incubation with 0.1% pepsin in 0.1M phosphate-buffered saline (room temperature, 10 minutes), rinsing twice in 0.1M phosphate-buffered saline (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 three times in phosphate-buffered saline (pH 7.4, 10 min each), incubation with secondary antibody (each in 1:100 phosphate-buffered saline, room temperature, 2 hours), rinsing four times in phosphate-buffered saline (10 minutes each), postfixation in 0.2% glutaraldehyde solution in 0.1M phosphate-buffered saline (5 min) and rinsing three times in phosphate-buffered saline (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. [4]
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Analysing morphologic features more detailed in both groups of lamellar macular hole and macular pseudohole each of 5 specimen 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 Zeiss light microscope and a Zeiss EM 9 S-2 electron microscope (Zeiss, Jena, Germany) five grids (each with six to nine ultrathin sections) per specimen have been imaged and evaluated by two experienced examiners. In five cases, more than three 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
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Clinical data analysis In this study, we included 25 eyes (11 lamellar macular holes and 14 macular pseudoholes) who 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. The Table 1 shows the clinical data of patients with lamellar macular hole and macular pseudohole including sex, age, diagnosis, metamorphopsia, preop and postop BCVA, preop and postop state of the lens and the period of follow-up in months. Before surgery, eyes with lamellar macular hole showed a median BCVA of LogMAR 0.40 (mean 0.43 ± 0.12 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 = 0.047). One of 11 patients with lamellar macular hole was found with unchanged BCVA, and two of 11 patients 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 = 0.010). 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 > 0.05). 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 [5]
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and intraocular lens implantation and 2 eyes with macular pseudohole underwent vitrectomy only. Overall, two 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, the remaining 3 eyes with lamellar macular hole 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.
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Spectral-domain optical coherence tomography analysis The 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; Figure 1, 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; Figure 2, Top right) Comparing these three groups of eyes with lamellar macular holes, there was no statistical difference in median BCVA (Mann Whitney Test, p > 0.05). In eyes with macular pseudohole, conventional epiretinal membranes were present in all examined eyes (100%). (Figure 3, Top left; Figure 3, Top right) They were identified as tractional epiretinal membrane with retinal folds and thickening of retinal layers. Lamellar macular hole-associated epiretinal proliferation was not seen in eyes with macular pseudoholes. Analysing the presence of vitreomacular adhesion, one eye with lamellar macular hole showed a foveal vitreomacular adhesion but none of the eyes with macular pseudoholes. Differentiating the state of posterior vitreous detachment into complete and partial by OCT, we found 3 eyes (2 eyes with lamellar macular holes and 1 eye with macular pseudohole) presenting with partial posterior vitreous detachment. All other eyes were seen with complete posterior vitreous detachment.
Cell density and cell distribution analysis Using interference and phase contrast microscopy, cell count and cell distribution were analysed in all specimens. The Table 2 shows cell distribution analysis of eyes with lamellar macular hole and macular pseudoholes. In specimens of eyes with lamellar macular hole, a total number of 1533 ± 1441 cells (range, 175-4942) were counted, presenting with a cell density of 295 ± 200 cells per mm2 (range, 91-714 cells per mm2). In 9 of 11 eyes, cell distribution was seen as a homogenous layer. In the 2 remaining eyes, clusters of cells were present. In the group of eyes with macular pseudohole, a total number of 2565 ± 4473 cells (range, 201-17771) were counted, showing a cell density of 348 ± 255 cells per mm2 (range, 13-853 cells per mm2). In 9 of 14 eyes, cell distribution was homogenous. In 4 of 14 eyes, clusters of cells were documented. Only, [6]
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one of the 14 specimens showed single cells distributed on the internal limiting membrane. There was no statistically difference in cell density between eyes with lamellar macular hole and macular pseudohole.
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Immunocytochemical analysis Analysis of flat-mounted specimens showed positive immunostaining for anti-glial fibrillary acidic protein and for the hyalocyte cell markers anti-CD45 and anti-CD64 in eyes with lamellar macular hole. (Figure 1, Middle left; Figure 1, Middle right) The Table 3 shows immunoreactivity of specimens removed from eyes with lamellar macular hole and macular pseudohole. Anti-laminin and anti-vimentin were also strongly positive as well as immunolabelling for anti-collagen type I and anti-collagen type III. In several specimens, anti-CD64, anti-collagen type I and anti-collagen type II was colocalized with anti-glial fibrillary acidic protein. In contrast, eyes with macular pseudohole presented a predominance of positive immunostaining for anti-α-smooth muscle actin, a marker for myofibroblast-like transdifferentation. (Figure 3, Middle left) Furthermore, anti-glial fibrillary acidic protein, the hyalocytes markers anti-CD45 and anti-CD64 as well as anti-collagen type II was frequently positive in eyes with macular pseudohole. (Figure 3, Middle right) A colocalization for anti-α-smooth muscle actin with CD45 was often found. In all control specimens, no specific positive immunostaining was observed.
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Ultrastructural analysis Using transmission electron microscopy, the internal limiting membrane was seen in all eyes in with macular pseudohole and in 9 of 11 specimens of eyes with lamellar macular hole. The Table 4 shows the ultrastrucutral features of specimens removed from eyes with lamellar macular hole and macular pseudohole. Characterized by its undulated retinal side and the smooth vitreal side, the internal limiting membrane was clearly differentiated from attached collagen strands. In eyes with lamellar macular hole, fibroblasts and hyalocytes were predominant. (Figure 1, Bottom left; Figure 1, Bottom right) Characterized by abundant rough endoplasmatic reticulum, prominent golgi complex and a fusiform shape of the cell body and nucleus, fibroblasts were frequently located in cell agglomerations. Cell agglomerations were seen in eyes with lamellar macular hole and presence of lamellar hole-associated epiretinal proliferation only, and consisted of masses of densely packed epiretinal cell proliferation. (Figure 2, Bottom right) Hyalocytes that can be distinguished by their lobulated cell nuclei, intracellular vacuoles, vesicles and mitochondria as well as long cell fibers were also located in cell agglomerations next to fibroblasts. However, in cell agglomerations hyalocytes were less frequently seen than fibroblasts. Myofibroblasts were present as well but sparsely seen in eyes with lamellar macular hole and lamellar hole-associated epiretinal proliferation compared to eyes with macular pseudohole. Myofibroblast-like cells contain cell fibers with contractile forces. In the extracellular matrix native vitreous collagen was identified as the major type of collagen. (Figure 2, Bottom left) It appeared in a regular arrangement of fibrils with a collagen fibril diameter of less than 16 nm. In several specimens, newly formed collagen with irregular fibril arrangement and fibril diameter of more than 16 nm was present as well as fibrous long spacing collagen. In some specimens, all three collagen types were [7]
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seen dispersed within cell proliferation. The latter was mostly seen surrounded by native vitreous collagen, characterized by a periodicity of approximately 100 nm. In contrast, eyes with macular pseudohole predominantly presented with myofibroblasts located in multilayered epiretinal proliferation. (Figure 3, Bottom left; Figure 3, Bottom right) Myofibroblastes are characterized by their aggregates of 5- to 7 nm subplasmalemmal cytoplasmatic filaments with fusiform densities, few rough endoplasmatic reticulum and long cell fibers with contractile properties. As a consequence of contractive cell properties, the internal limiting membrane was mostly multiply folded by cell proliferation spanning from one fold to the other. Native vitreous collagen was frequently embedded as continuous layer between the epiretinal cells and the internal limiting membrane. Fibrous long spacing collagen was seen more frequently in eyes with macular pseudohole than in eyes with lamellar macular holes, but exclusively embedded in native vitreous collagen. (Figure 3, Bottom right) Newly formed collagen was occasionally seen.
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DISCUSSION The herein presented immunocytochemical and ultrastructural study was conducted to compare epiretinal tissue removed from eyes with lamellar macular hole and macular pseudohole. We analysed specimens harvested by sequential epiretinal membrane and internal limiting membrane peeling during vitrectomy. Based on Spectral-domain optical coherence tomography findings, the removed epiretinal tissue was retrospectively differentiated into lamellar hole-associated epiretinal proliferation and conventional epiretinal membrane. The terminology used in this study was recently suggested by Pang and colleagues since a widely accepted term for lamellar hole-associated epiretinal proliferation was lacking.7-8 On Spectral-domain optical coherence tomography, lamellar hole-associated epiretinal proliferation is characterized by a homogenous appearance without any contractive properties, and it can clearly be distinguished from conventional epiretinal membrane that is seen as a thin hyper-reflective layer of tractional epiretinal tissue on the retinal surface. Although, lamellar hole-associated epiretinal proliferation was mostly found in lamellar macular hole it can occasionally be identified in other retinal diseases such as full-thickness macular holes.7-8 In this study, lamellar hole-associated epiretinal proliferation was demonstrated in eight of eleven eyes (73%) with lamellar macular hole and in none of the eyes with macular pseudohole. In five of these eyes, a combination of both lamellar holeassociated epiretinal proliferation and conventional contractive epiretinal membrane was found on Spectral-domain optical coherence tomography. Differences in morphology of epiretinal tissue in lamellar macular holes have also been reported in recent Spectral-domain optical coherence tomography studies showing that eyes with lamellar macular hole and lamellar hole-associated epiretinal proliferation were correlated with photoreceptor layer defects and worse visual acuity compared to eyes with lamellar macular hole and contractive conventional epiretinal membrane.6,8 Conventional epiretinal membranes exerting traction onto retinal layers are well known and occur in different traction maculopathies such as macular pucker with or without macular pseudohole, idiopathic full-thickness macular holes and vitreomacular traction syndrome. In macular pseudoholes, the presence of epiretinal membrane is given by [8]
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definition, whereas the presence of epiretinal tissue in lamellar macular holes was first described after introduction of high resolution OCT technology.2-3,10-12 Although important advances of OCT technology were made during recent years, there is little detail on cell and collagen composition in lamellar hole-associated epiretinal proliferation. In contrast, conventional epiretinal membranes are immunocytochemically and ultrastructurally well described.13-14 In this study, we demonstrated positive immunolabelling of collagen type I, II and III in specimens removed from eyes with lamellar macular hole. Anti-collagen type I and III are targeting newly formed collagen, whereas anti-collagen type II represents vitreous collagen. Immunolabelling for vimentin and glial fibrillary acidic protein and immunolabelling for CD45 and CD64 were positive in eyes with lamellar hole-associated epiretinal proliferation that may show presence of glial cells and hyalocytes, respectively. However, beside glial cell, hyalocytes and fibroblasts were reported to be seen with positive immunoreactivity for anti- glial fibrillary acidic protein as well.15 In contrast, specimens of conventional epiretinal membranes of macular pseudoholes demonstrated a positive immunolabelling for α-smooth muscle actin and collagen type I which distinguished them decisively from lamellar hole-associated epiretinal proliferation of lamellar macular hole. As an intracellular actin filament, αsmooth muscle actin is suggested to be essential for extracellular matrix contraction in co-localization with collagen type I.16-17 This supports the hypothesis of myofibroblastlike transdifferentiation. Cells of glial origin are presumed to lose their typical properties in gain of contractile features.18 This immunocytochemical finding of conventional epiretinal membrane has already been shown in other vitreoretinal diseases such as full-thickness macular hole and macular traction syndrome, and it represents more potential of tractional force generation compared to lamellar hole-associated epiretinal proliferation of lamellar macular holes.13,15 By transmission electron microscopy, we found a predominance of fibroblasts and hyalocytes in eyes with lamellar hole-associated epiretinal proliferation. The occurrence of cell agglomeration was a typical feature in these specimens. In lamellar hole-associated epiretinal proliferation, masses of cells were located very close to each other, and native vitreous collagen was either dispersed in cell proliferations or seen as thick undulated strands of collagen. Furthermore, fibrous long spacing collagen was found in eyes with lamellar macular hole and in eyes with macular pseudoholes. As a fibrillar precipitate of collagen molecules, fibrous long spacing collagen is typically seen embedded in native vitreous collagen. Being an intermediate stage of collagen in the degradation of normal fibrillar collagen, fibrous long spacing collagen is believed to represent a remodelling process of premacular vitreous cortex.19-20 Cell-cell and cellmatrix interactions appear to cause structural changes in collagen which may lead to diffusely condensed cortical vitreous. Fibrous long spacing collagen was recently reported to be found in lamellar hole-associated epiretinal proliferation by Parolini and colleagues who used the term ‘atypical epiretinal tissue’ to describe lamellar holeassociated epiretinal proliferation.3 In conventional epiretinal membranes of eyes with macular pseudoholes, myofibroblasts were found as predominant cell type by transmission electron microscopy. Located in multilayers, myofibroblasts were seen tightly packed over internal limiting membrane folds in correlation to Spectral-domain optical coherence [9]
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tomography findings of conventional epiretinal membranes. Thick native vitreous collagen strands and fibrous long spacing collagen were present as well which points to an important role of vitreous cortex collagen in the pathogenesis of traction related conventional epiretinal membranes. Given the differences in cell type and cell distribution of epiretinal tissue, we hypothesize that pathogenesis may differ in subgroups of eyes with lamellar macular hole and macular pseudoholes. This is in accordance to several clinical studies reporting on different clinical course and response to surgery in these eyes. However, there are certain characteristics that both entities seem to have in common, such as immunoreactivity for glial cell markers and hyalocyte cell markers as well as presence and distribution of native vitreous collagen and fibrous long spacing collagen. This study has some limitations mainly based on its small case series and short period of follow-up examinations. Therefore, we did not focus on statistical analysis and correlation of structural findings with functional parameters. In summary, lamellar hole-associated epiretinal proliferation mainly differs from conventional epiretinal membrane of macular pseudohole in cell type and cell distribution pointing to differences in pathogenesis that might explain different clinical course and response to surgery in these eyes. In lamellar hole-associated epiretinal proliferation, fibroblast and hyalocytes are predominant and located in dense cell agglomerations without contractive components, whereas myofibroblasts are the predominant cell type in conventional epiretinal membrane that are located in cell multilayers tightly stretched over folds of internal limiting membrane or native vitreous collagen. We conclude that cells within lamellar hole-associated epiretinal proliferation appear to originate from vitreous and possess less contractive properties than cells of conventional epiretinal membrane. Of note, we found eyes with both lamellar holeassociated epiretinal proliferation and conventional epiretinal membrane in lamellar macular hole that were also detectable on Spectral-domain optical coherence tomography examination. Therefore, it is of particular importance to analyse all OCT scans of each examination of eyes with lamellar macular hole. Based on high resolution OCT analysis, future studies may help to define subgroups of lamellar macular hole that are eligible for macular surgery by differentiation of lamellar hole-associated epiretinal proliferation from contractive epiretinal membranes.
ACKNOWLEDGMENT ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST. The authors indicate no financial conflict of interest involved in design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, and approval of the manuscript. This study was supported by unrestricted grants of the Ludwig-Maximilians-University, Munich, Germany, Grant for Research and Education (FöFoLe, ID # 822). Involved in design and conduct of study (D.C., R.G.S.); collection, management, analysis and interpretation of data (D.C., C.H., A.W., R.G.S.); specimen preparation (D.C., E.E., R.S.) and critical review and approval (D.C., C.H., W.J.M., A.W., A.K., R.G.S.) of manuscript. [10]
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REFERENCES 1. Haouchine B, Massin P, Tadayoni R, Erginay A, Gaudric A. Diagnosis of macular pseudoholes and lamellar macular holes by optical coherence tomography. Am J Ophthalmol 2004;138(5):732-739.
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2. Witkin AJ, Ko TH, Fujimoto JG, et al. Redefining lamellar holes and the vitreomacular interface: an ultrahigh-resolution optical coherence tomography study. Ophthalmology 2006;113(3):388-397.
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3. Parolini B, Schumann RG, Cereda MG, Haritoglou C, Pertile G. Lamellar macular hole: a clinicopathologic correlation of surgically excised epiretinal membranes. Invest Ophthalmol Vis Sci 2011;52(12):9074-9083.
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4. Michalewska Z, Michalewski J, Odrobina D, Nawrocki J. Non-full-thickness macular holes reassessed with spectral domain optical coherence tomography. Retina 2012;32(5):922-929. 5. Bottoni F, Deiro AP, Giani A, Orini C, Cigada M, Staurenghi G. The natural history of lamellar macular holes: a spectral domain optical coherence tomography study. Graefes Arch Clin Exp Ophthalmol 2013;251(2):467-475.
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6. Schumann RG, Compera D, Schaumberger MM, et al. Epiretinal membrane characteristics correlate with photoreceptor layer defects in lamellar macular holes and macular pseudoholes. Retina 2015;35(4):727-735. 7. Pang CE, Spaide RF, Freund KB. Epiretinal proliferation seen in association with lamellar macular holes: a distinct clinical entity. Retina 2014;34(8):1513-1523.
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8. Pang CE, Spaide RF, Freund KB. Comparing functional and morphologic characteristics of lamellar macular holes with and without lamellar hole-associated epiretinal proliferation. Retina 2015;35(4):720-726.
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9. Duker JS, Kaiser PK, Binder S, et al. The International Vitreomacular Traction Study Group classification of vitreomacular adhesion, traction, and macular hole. Ophthalmology 2013;120(12):2611-2619. 10. Gass JD. Lamellar macular hole: a complication of cystoid macular edema after cataract extraction: a clinicopathologic case report. Trans Am Ophthalmol Soc 1975;73:231-250. 11. Theodossiadis PG, Grigoropoulos VG, Emfietzoglou I, et al. Evolution of lamellar macular hole studied by optical coherence tomography. Graefes Arch Clin Exp Ophthalmol 2009;247(1):13-20. 12. Androudi S, Stangos A, Brazitikos PD. Lamellar macular holes: tomographic features and surgical outcome. Am J Ophthalmol 2009;148(3):420-426. [11]
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13. Zhao F, Gandorfer A, Haritoglou C, et al. Epiretinal cell proliferation in macular pucker and vitreomacular traction syndrome: analysis of flat-mounted internal limiting membrane specimens. Retina 2013;33(1):77-88.
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14. Schumann RG, Schaumberger MM, Rohleder M, Haritoglou C, Kampik A, Gandorfer A. Ultrastructure of the vitreomacular interface in full-thickness idiopathic macular holes: a consecutive analysis of 100 cases. Am J Ophthalmol 2006;141(6):1112-1119.
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15. Schumann RG, Eibl KH, Zhao F, et al. Immunocytochemical and ultrastructural evidence of glial cells and hyalocytes in internal limiting membrane specimens of idiopathic macular holes. Invest Ophthalmol Vis Sci 2011;52(11):7822-7834.
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16. Arora PD, McCulloch CA. Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts. J Cell Physiol 1994;159(1):161-175. 17. Hinz B. It has to be the αv: myofibroblast integrins activate latent TGF-beta1. Nat Med 2013;19(12):1567-1568. 18. Bringmann A, Wiedemann P. Involvement of Muller glial cells in epiretinal membrane formation. Graefes Arch Clin Exp Ophthalmol 2009;247(7):865-883.
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19. Dingemans KP, Teeling P. Long-spacing collagen and proteoglycans in pathologic tissues. Ultrastruct Pathol 1994;18(6):539-547.
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20. Ishida S, Yamazaki K, Shinoda K, Kawashima S, Oguchi Y. Macular hole retinal detachment in highly myopic eyes: ultrastructure of surgically removed epiretinal membrane and clinicopathologic correlation. Retina 2000;20(2):176-183.
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FIGURE LEGENDS
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Figure 1. Lamellar hole-associated epiretinal proliferation. (Top left, Top right) Spectraldomain optical coherence tomography images of a 70-years old female presenting lamellar macular hole with lamellar hole-associated epiretinal proliferation (white arrowhead). (Middle left) Immunolabelling and cell nuclei staining (blue) of flat-mounted specimen demonstrate positive immunoreactivity for the hyalocyte cell marker antiCD64 (red) and (Middle right) positive immunoreactivity for the glial cell marker anti-glial fibrillary acidic protein (green). (Bottom left) Transmission electron micrograph of lamellar hole-associated epiretinal proliferation shows abundant cell agglomeration of fibroblasts and hyalocytes. (Bottom right) Rectangle of (Bottom left) in detail, representing fibroblasts characterized by abundant rough endoplasmatic reticulum (black arrow). (Original magnification: (C) x100; (D) x400; (E) x3,000; (F) x120,000)
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Figure 2. Combination of both lamellar hole-associated epiretinal proliferation and conventional epiretinal membrane. (Top left, Top right) Spectral-domain optical coherence tomography images of a 79-years old male with lamellar macular hole associated with both a conventional epiretinal membrane (white arrow) and lamellar hole-associated epiretinal proliferation (white arrowhead). (Middle left) Light micrograph with toluidine blue staining of serial semi-thin sections shows a compact and tightly stretched epiretinal membrane on folded internal limiting membrane. (Middle right) Light micrograph demonstrates abundant epiretinal cell proliferation embedded in extracellular matrix of a specimen with lamellar hole-associated epiretinal proliferation. (Bottom left) Transmission electron micrograph shows myofibroblasts of conventional epiretinal membrane located on folded vitreous collagen strand (NVC) (black arrow). (Bottom right) Lamellar hole-associated epiretinal proliferation was seen as compact cell agglomeration with abundant fibroblasts. (Original magnification: (C, D) x200; (E) x1,800, (F) x1,800)
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Figure 3. Conventional epiretinal membrane of macular pseudohole. (Top left, Top right) Spectral-domain optical coherence tomography images of a 72-years old male with macular pseudohole and conventional epiretinal membrane (white arrow). (Middle left) Immunolabelling and cell nuclei staining (blue) of flat-mounted specimen demonstrate positive immunoreactivity for the myofibroblast cell marker anti-α-smooth muscle actin (red) and (Middle right) positive immunoreactivity for the glial cell marker anti-glial fibrillary acidic protein (green). (Bottom left) Transmission electron micrograph of conventional epiretinal membrane shows cell multilayer with myofibroblasts located on native vitreous collagen on the vitreal side of the internal limiting membrane (asterisk). (Bottom right) Transmission electron micrograph representing myofibroblast located on a thick vitreous cortex collagen strand with embedded fibrous long spacing collagen (detail). (Original magnification: (C, D) x100; (E) x3,000; (F) x4,400)
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Meta-
Sex/Age
Diagnosis
morphopsia
Preop BCVA
Postop BCVA
Preop state
Postop state
Follow-up
(+/-)
(LogMAR)
(LogMAR)
of the lens
of the lens
(months)
IOL
21
IOL
18
M/74
LMH
-
0.5
0.1
Phakic
2
M/71
LMH
+
0.3
0.4
Phakic
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
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Table 1. Clinical data of patients with lamellar macular hole and macular pseudohole.
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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
23
F/64
MPH
-
0.2
0.2
Phakic
24
M/77
MPH
-
0.4
0.4
Phakic
25
F/72
MPH
-
0.2
0.2
Phakic
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IOL
4
IOL
3
IOL
4
IOL
3
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M, male; F, female; LMH, lamellar macular hole; MPH, macular pseudohole; BCVA, bestcorrected visual acuity; IOL, intraocular lens.
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Table 2. Spectral-domain optical coherence tomography analysis and cell distribution analysis of specimens removed from eyes with lamellar macular holes and macular pseudoholes. SD-OCT analysis
Area of
peeled
State of posterior hyaloid
ERM
No of
cells
ILM
LHEP
2
(mm ) LMH
complete PVD
x
-
6.9
2246
2
LMH
complete PVD
x
x
13.32
4942
3
LMH
complete PVD
x
-
15.44
4
LMH
complete PVD
x
x
5
LMH
partial PVD
-
x
6
LMH
complete PVD
x
x
7
LMH
complete PVD
x
-
8
LMH
partial PVD
-
9
LMH
complete PVD
10
LMH
complete PVD
11
LMH
complete PVD
(cells/mm )
Single cells
Homogenous
Cluster of cells
layer of cells
326
-
x
-
371
-
x
-
2651
172
-
x
-
3.62
2583
714
-
x
-
6.74
779
116
-
x
-
7.06
867
123
-
x
-
1.63
405
248
-
x
-
1.93
175
91
-
-
x
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x
Cell distribution
2
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Cell density
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No
-
x
2.27
322
142
-
-
x
x
x
1.81
995
550
-
x
-
x
x
2.24
898
401
-
x
-
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MPH
complete PVD
x
-
34.86
17771
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
25
MPH
complete PVD
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-
x
-
4.09
889
217
x
-
-
x
-
4.2
1669
397
-
x
-
LMH, lamellar macular hole; MPH, macular pseudohole; PVD, posterior vitreous detachment; SD-OCT, Spectral-domain optical coherence tomography; ERM, epiretinal membrane; LHEP, lamellar hole-associated epiretinal proliferation; ILM, internal limiting membrane.
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Anti-
Target structure
Immunoreactivity LMH
MPH
(N = 11)
(N = 14)
++
+
Intermediate type
(GFAP)
filaments of glial cells
Vimentin
Mueller cells, astrocytes
++
(+)
Alpha- smooth muscle actin (α-
Intracellular actin
(+)
++
SMA)
filaments
CD 45
Hyalocytes
++
+
CD 64
Hyalocytes
++
+
Collagen type I
Newly formed collagen
++
+
Collagen type II
Vitreous cortex collagen
+
(+)
Collagen type III
Extracelluar matrix
++
(+)
Laminin
Basalmembrane-ILM
++
++
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Glial fibrillary acidic protein
LMH, lamellar macular hole; MPH, macular pseudohole; ILM, internal limiting membrane.
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++, very present; +, present; (+), sparsely present; -, absent.
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Ultrastructural features
LMH (N = 11 ) 9
MPH (N = 14 ) 14
Single cells
0
1
Monolayer of cells
1
1
Multilayer of cells
3
12
Cell agglomeration
7
0
Hyalocytes
6
8
Macrophages
0
Fibrous astrocytes
1
Myofibroblasts
4
Inner limiting membrane
1 1
12
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Fibroblasts
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Cell distribution at the ILM
8
3
0
1
1
7
4
12
6
4
Native vitreous collagen
9
14
Newly formed collagen
6
7
Fibrous long spacing collagen
3
7
Retinal pigment epithelial cells Collagen distribution Thick vitreous collagen strands Vitreous collagen on ILM Collagen dispersed with cells
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Collagen types
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LMH, lamellar macular hole; MPH, macular pseudohole; ILM, inner limiting membrane.
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Denise Compera, MD, is currently undergoing her residency at the Department of Ophthalmology, Ludwig-Maximilians-University Munich, Germany. Since 2012, she joined the research group of “Vitreoretinal pathology and electron microscopy“. Dr Compera’s research interests include diseases of the vitreoretinal interface, in particular imaging techniques and surgical intervention in traction maculopathies.
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S0002-9394(15)00277-9
DOI:
10.1016/j.ajo.2015.05.010
Reference:
AJOPHT 9331
To appear in:
American Journal of Ophthalmology
Received Date: 9 March 2015 Revised Date:
11 May 2015
Accepted Date: 12 May 2015
Please cite this article as: Compera D, Entchev E, Haritoglou C, Scheler R, Mayer WJ, Wolf A, Kampik A, Schumann RG, Lamellar hole - associated epiretinal proliferation in comparison to epiretinal membranes of macular pseudoholes, American Journal of Ophthalmology (2015), doi: 10.1016/ j.ajo.2015.05.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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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 analysed 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.
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Lamellar hole - associated epiretinal proliferation in comparison to epiretinal membranes of macular pseudoholes
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Denise Compera, MD; Enrico Entchev, MD; Christos Haritoglou, MD; Renate Scheler, MTA; Wolfgang J Mayer, MD; Armin Wolf, MD; Anselm Kampik, MD; Ricarda G Schumann, MD Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany
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Short Title: Lamellar hole-associated epiretinal proliferation
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Correspondence to:
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Denise Compera, MD Ludwig-Maximilians-University, Department of Ophthalmology, Mathildenstrasse 8, 80336 Munich, Germany Tel.: +49-89-5160 3811 Fax: +49-89-5160 5160 Email: [email protected]
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INTRODUCTION 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.1-8 First described by Witkin et al., 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 fibre layer on OCT images.2 Parolini et al. also reported on unusual epiretinal proliferation in eyes with lamellar macular hole in a clinical-pathological case series, and named it “dense” membranes that differ from “tractional” membranes according to their morphologic features.3 Since there is no widely accepted terminology, Pang et al. 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.7-8 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.6-8 According to our current knowledge of the pathogenesis of vitreo-maculopathies, 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.3,6-8 In eyes with macular pseudoholes, generation of tractional forces by conventional epiretinal membranes become 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 two 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.5,6,11 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.7 This hypothesis is in accordance to histopathological findings of Parolini et al. 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 in contrast to conventional epiretinal membranes in macular pseudoholes. Therefore, the aim of this study was to analyse 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 clinical-pathological 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 two surgeons and prepared for flat-mount preparation, [2]
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immunocytochemistry, phase-contrast, 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, gender, preoperative BCVA, postoperative BCVA, period of follow-up, pre- and postoperative state of the lens, as well as 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 histopathological 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.
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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-scans of baseline visits. According to their appearance and reflectivity, epiretinal membranes were classified by Spectral-domain optical coherence tomography as previously published.1-2,9 Epiretinal membranes with contractive properties were termed ‘conventional epiretinal membrane’. In Spectral-domain optical coherence tomography, they appear as a thin hyper-reflective layer exerting traction on the inner retinal layers represented as visible retinal folds. In accordance to Pang et al., epiretinal material of homogenous medium reflectivity without any contractive properties on the retinal surface was termed ‘lamellar hole-associated epiretinal proliferation’.7-8 The presence of vitreomacular adhesion and the state of posterior vitreous detachment was analysed by reviewing all OCT scans from each examination.
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Surgical procedure Patients were recommended to surgery according to the following indication criteria: (1) best-corrected visual acuity (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 detaching 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 endgripping 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 one 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 being not easy to grasp. The vitreous cavity was filled with a tamponade of either 15% hexafluoroethane (C2F6) gasair mixture, or air, or balanced salt solution. Individually, patients were instructed to keep a face-down position for two days. [3]
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Immunocytochemistry On average, two specimens (range 1-4) were harvested from each patient. There was no selection of specimens before the preparation procedure. If more than two specimens were removed from one eye, all specimens were prepared and analysed. If less than three specimens were removed, large specimens were segmented for labelling combinations of all nine 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 x50 and x400. For photo 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 antiCD64, 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 anticollagen type III, Biotrend, Cologne, Germany). Manufacturer`s instructions were followed exactly. The specimens were labelled with combinations of three primary antibodies. As secondary antibody we used either donkey anti-rabbit Cy2, donkey antimouse Cy3, or donkey anti-mouse Cy5 (Dianova, Hamburg, Germany). Indirect immunocytochemistry comprised the following steps: rinsing in 0.1M phosphate-buffered saline (PBS, pH 7.4), incubation with 0.1% pepsin in 0.1M phosphate-buffered saline (room temperature, 10 minutes), rinsing twice in 0.1M phosphate-buffered saline (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 three times in phosphate-buffered saline (pH 7.4, 10 min each), incubation with secondary antibody (each in 1:100 phosphate-buffered saline, room temperature, 2 hours), rinsing four times in phosphate-buffered saline (10 minutes each), postfixation in 0.2% glutaraldehyde solution in 0.1M phosphate-buffered saline (5 min) and rinsing three times in phosphate-buffered saline (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. [4]
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Analysing morphologic features more detailed in both groups of lamellar macular hole and macular pseudohole each of 5 specimen 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 Zeiss light microscope and a Zeiss EM 9 S-2 electron microscope (Zeiss, Jena, Germany) five grids (each with six to nine ultrathin sections) per specimen have been imaged and evaluated by two experienced examiners. In five cases, more than three 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
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Clinical data analysis In this study, we included 25 eyes (11 lamellar macular holes and 14 macular pseudoholes) who 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. The Table 1 shows the clinical data of patients with lamellar macular hole and macular pseudohole including sex, age, diagnosis, metamorphopsia, preop and postop BCVA, preop and postop state of the lens and the period of follow-up in months. Before surgery, eyes with lamellar macular hole showed a median BCVA of LogMAR 0.40 (mean 0.43 ± 0.12 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 = 0.047). One of 11 patients with lamellar macular hole was found with unchanged BCVA, and two of 11 patients 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 = 0.010). 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 > 0.05). 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 [5]
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and intraocular lens implantation and 2 eyes with macular pseudohole underwent vitrectomy only. Overall, two 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, the remaining 3 eyes with lamellar macular hole 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.
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Spectral-domain optical coherence tomography analysis The 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; Figure 1, 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; Figure 2, Top right) Comparing these three groups of eyes with lamellar macular holes, there was no statistical difference in median BCVA (Mann Whitney Test, p > 0.05). In eyes with macular pseudohole, conventional epiretinal membranes were present in all examined eyes (100%). (Figure 3, Top left; Figure 3, Top right) They were identified as tractional epiretinal membrane with retinal folds and thickening of retinal layers. Lamellar macular hole-associated epiretinal proliferation was not seen in eyes with macular pseudoholes. Analysing the presence of vitreomacular adhesion, one eye with lamellar macular hole showed a foveal vitreomacular adhesion but none of the eyes with macular pseudoholes. Differentiating the state of posterior vitreous detachment into complete and partial by OCT, we found 3 eyes (2 eyes with lamellar macular holes and 1 eye with macular pseudohole) presenting with partial posterior vitreous detachment. All other eyes were seen with complete posterior vitreous detachment.
Cell density and cell distribution analysis Using interference and phase contrast microscopy, cell count and cell distribution were analysed in all specimens. The Table 2 shows cell distribution analysis of eyes with lamellar macular hole and macular pseudoholes. In specimens of eyes with lamellar macular hole, a total number of 1533 ± 1441 cells (range, 175-4942) were counted, presenting with a cell density of 295 ± 200 cells per mm2 (range, 91-714 cells per mm2). In 9 of 11 eyes, cell distribution was seen as a homogenous layer. In the 2 remaining eyes, clusters of cells were present. In the group of eyes with macular pseudohole, a total number of 2565 ± 4473 cells (range, 201-17771) were counted, showing a cell density of 348 ± 255 cells per mm2 (range, 13-853 cells per mm2). In 9 of 14 eyes, cell distribution was homogenous. In 4 of 14 eyes, clusters of cells were documented. Only, [6]
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one of the 14 specimens showed single cells distributed on the internal limiting membrane. There was no statistically difference in cell density between eyes with lamellar macular hole and macular pseudohole.
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Immunocytochemical analysis Analysis of flat-mounted specimens showed positive immunostaining for anti-glial fibrillary acidic protein and for the hyalocyte cell markers anti-CD45 and anti-CD64 in eyes with lamellar macular hole. (Figure 1, Middle left; Figure 1, Middle right) The Table 3 shows immunoreactivity of specimens removed from eyes with lamellar macular hole and macular pseudohole. Anti-laminin and anti-vimentin were also strongly positive as well as immunolabelling for anti-collagen type I and anti-collagen type III. In several specimens, anti-CD64, anti-collagen type I and anti-collagen type II was colocalized with anti-glial fibrillary acidic protein. In contrast, eyes with macular pseudohole presented a predominance of positive immunostaining for anti-α-smooth muscle actin, a marker for myofibroblast-like transdifferentation. (Figure 3, Middle left) Furthermore, anti-glial fibrillary acidic protein, the hyalocytes markers anti-CD45 and anti-CD64 as well as anti-collagen type II was frequently positive in eyes with macular pseudohole. (Figure 3, Middle right) A colocalization for anti-α-smooth muscle actin with CD45 was often found. In all control specimens, no specific positive immunostaining was observed.
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Ultrastructural analysis Using transmission electron microscopy, the internal limiting membrane was seen in all eyes in with macular pseudohole and in 9 of 11 specimens of eyes with lamellar macular hole. The Table 4 shows the ultrastrucutral features of specimens removed from eyes with lamellar macular hole and macular pseudohole. Characterized by its undulated retinal side and the smooth vitreal side, the internal limiting membrane was clearly differentiated from attached collagen strands. In eyes with lamellar macular hole, fibroblasts and hyalocytes were predominant. (Figure 1, Bottom left; Figure 1, Bottom right) Characterized by abundant rough endoplasmatic reticulum, prominent golgi complex and a fusiform shape of the cell body and nucleus, fibroblasts were frequently located in cell agglomerations. Cell agglomerations were seen in eyes with lamellar macular hole and presence of lamellar hole-associated epiretinal proliferation only, and consisted of masses of densely packed epiretinal cell proliferation. (Figure 2, Bottom right) Hyalocytes that can be distinguished by their lobulated cell nuclei, intracellular vacuoles, vesicles and mitochondria as well as long cell fibers were also located in cell agglomerations next to fibroblasts. However, in cell agglomerations hyalocytes were less frequently seen than fibroblasts. Myofibroblasts were present as well but sparsely seen in eyes with lamellar macular hole and lamellar hole-associated epiretinal proliferation compared to eyes with macular pseudohole. Myofibroblast-like cells contain cell fibers with contractile forces. In the extracellular matrix native vitreous collagen was identified as the major type of collagen. (Figure 2, Bottom left) It appeared in a regular arrangement of fibrils with a collagen fibril diameter of less than 16 nm. In several specimens, newly formed collagen with irregular fibril arrangement and fibril diameter of more than 16 nm was present as well as fibrous long spacing collagen. In some specimens, all three collagen types were [7]
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seen dispersed within cell proliferation. The latter was mostly seen surrounded by native vitreous collagen, characterized by a periodicity of approximately 100 nm. In contrast, eyes with macular pseudohole predominantly presented with myofibroblasts located in multilayered epiretinal proliferation. (Figure 3, Bottom left; Figure 3, Bottom right) Myofibroblastes are characterized by their aggregates of 5- to 7 nm subplasmalemmal cytoplasmatic filaments with fusiform densities, few rough endoplasmatic reticulum and long cell fibers with contractile properties. As a consequence of contractive cell properties, the internal limiting membrane was mostly multiply folded by cell proliferation spanning from one fold to the other. Native vitreous collagen was frequently embedded as continuous layer between the epiretinal cells and the internal limiting membrane. Fibrous long spacing collagen was seen more frequently in eyes with macular pseudohole than in eyes with lamellar macular holes, but exclusively embedded in native vitreous collagen. (Figure 3, Bottom right) Newly formed collagen was occasionally seen.
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DISCUSSION The herein presented immunocytochemical and ultrastructural study was conducted to compare epiretinal tissue removed from eyes with lamellar macular hole and macular pseudohole. We analysed specimens harvested by sequential epiretinal membrane and internal limiting membrane peeling during vitrectomy. Based on Spectral-domain optical coherence tomography findings, the removed epiretinal tissue was retrospectively differentiated into lamellar hole-associated epiretinal proliferation and conventional epiretinal membrane. The terminology used in this study was recently suggested by Pang and colleagues since a widely accepted term for lamellar hole-associated epiretinal proliferation was lacking.7-8 On Spectral-domain optical coherence tomography, lamellar hole-associated epiretinal proliferation is characterized by a homogenous appearance without any contractive properties, and it can clearly be distinguished from conventional epiretinal membrane that is seen as a thin hyper-reflective layer of tractional epiretinal tissue on the retinal surface. Although, lamellar hole-associated epiretinal proliferation was mostly found in lamellar macular hole it can occasionally be identified in other retinal diseases such as full-thickness macular holes.7-8 In this study, lamellar hole-associated epiretinal proliferation was demonstrated in eight of eleven eyes (73%) with lamellar macular hole and in none of the eyes with macular pseudohole. In five of these eyes, a combination of both lamellar holeassociated epiretinal proliferation and conventional contractive epiretinal membrane was found on Spectral-domain optical coherence tomography. Differences in morphology of epiretinal tissue in lamellar macular holes have also been reported in recent Spectral-domain optical coherence tomography studies showing that eyes with lamellar macular hole and lamellar hole-associated epiretinal proliferation were correlated with photoreceptor layer defects and worse visual acuity compared to eyes with lamellar macular hole and contractive conventional epiretinal membrane.6,8 Conventional epiretinal membranes exerting traction onto retinal layers are well known and occur in different traction maculopathies such as macular pucker with or without macular pseudohole, idiopathic full-thickness macular holes and vitreomacular traction syndrome. In macular pseudoholes, the presence of epiretinal membrane is given by [8]
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definition, whereas the presence of epiretinal tissue in lamellar macular holes was first described after introduction of high resolution OCT technology.2-3,10-12 Although important advances of OCT technology were made during recent years, there is little detail on cell and collagen composition in lamellar hole-associated epiretinal proliferation. In contrast, conventional epiretinal membranes are immunocytochemically and ultrastructurally well described.13-14 In this study, we demonstrated positive immunolabelling of collagen type I, II and III in specimens removed from eyes with lamellar macular hole. Anti-collagen type I and III are targeting newly formed collagen, whereas anti-collagen type II represents vitreous collagen. Immunolabelling for vimentin and glial fibrillary acidic protein and immunolabelling for CD45 and CD64 were positive in eyes with lamellar hole-associated epiretinal proliferation that may show presence of glial cells and hyalocytes, respectively. However, beside glial cell, hyalocytes and fibroblasts were reported to be seen with positive immunoreactivity for anti- glial fibrillary acidic protein as well.15 In contrast, specimens of conventional epiretinal membranes of macular pseudoholes demonstrated a positive immunolabelling for α-smooth muscle actin and collagen type I which distinguished them decisively from lamellar hole-associated epiretinal proliferation of lamellar macular hole. As an intracellular actin filament, αsmooth muscle actin is suggested to be essential for extracellular matrix contraction in co-localization with collagen type I.16-17 This supports the hypothesis of myofibroblastlike transdifferentiation. Cells of glial origin are presumed to lose their typical properties in gain of contractile features.18 This immunocytochemical finding of conventional epiretinal membrane has already been shown in other vitreoretinal diseases such as full-thickness macular hole and macular traction syndrome, and it represents more potential of tractional force generation compared to lamellar hole-associated epiretinal proliferation of lamellar macular holes.13,15 By transmission electron microscopy, we found a predominance of fibroblasts and hyalocytes in eyes with lamellar hole-associated epiretinal proliferation. The occurrence of cell agglomeration was a typical feature in these specimens. In lamellar hole-associated epiretinal proliferation, masses of cells were located very close to each other, and native vitreous collagen was either dispersed in cell proliferations or seen as thick undulated strands of collagen. Furthermore, fibrous long spacing collagen was found in eyes with lamellar macular hole and in eyes with macular pseudoholes. As a fibrillar precipitate of collagen molecules, fibrous long spacing collagen is typically seen embedded in native vitreous collagen. Being an intermediate stage of collagen in the degradation of normal fibrillar collagen, fibrous long spacing collagen is believed to represent a remodelling process of premacular vitreous cortex.19-20 Cell-cell and cellmatrix interactions appear to cause structural changes in collagen which may lead to diffusely condensed cortical vitreous. Fibrous long spacing collagen was recently reported to be found in lamellar hole-associated epiretinal proliferation by Parolini and colleagues who used the term ‘atypical epiretinal tissue’ to describe lamellar holeassociated epiretinal proliferation.3 In conventional epiretinal membranes of eyes with macular pseudoholes, myofibroblasts were found as predominant cell type by transmission electron microscopy. Located in multilayers, myofibroblasts were seen tightly packed over internal limiting membrane folds in correlation to Spectral-domain optical coherence [9]
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tomography findings of conventional epiretinal membranes. Thick native vitreous collagen strands and fibrous long spacing collagen were present as well which points to an important role of vitreous cortex collagen in the pathogenesis of traction related conventional epiretinal membranes. Given the differences in cell type and cell distribution of epiretinal tissue, we hypothesize that pathogenesis may differ in subgroups of eyes with lamellar macular hole and macular pseudoholes. This is in accordance to several clinical studies reporting on different clinical course and response to surgery in these eyes. However, there are certain characteristics that both entities seem to have in common, such as immunoreactivity for glial cell markers and hyalocyte cell markers as well as presence and distribution of native vitreous collagen and fibrous long spacing collagen. This study has some limitations mainly based on its small case series and short period of follow-up examinations. Therefore, we did not focus on statistical analysis and correlation of structural findings with functional parameters. In summary, lamellar hole-associated epiretinal proliferation mainly differs from conventional epiretinal membrane of macular pseudohole in cell type and cell distribution pointing to differences in pathogenesis that might explain different clinical course and response to surgery in these eyes. In lamellar hole-associated epiretinal proliferation, fibroblast and hyalocytes are predominant and located in dense cell agglomerations without contractive components, whereas myofibroblasts are the predominant cell type in conventional epiretinal membrane that are located in cell multilayers tightly stretched over folds of internal limiting membrane or native vitreous collagen. We conclude that cells within lamellar hole-associated epiretinal proliferation appear to originate from vitreous and possess less contractive properties than cells of conventional epiretinal membrane. Of note, we found eyes with both lamellar holeassociated epiretinal proliferation and conventional epiretinal membrane in lamellar macular hole that were also detectable on Spectral-domain optical coherence tomography examination. Therefore, it is of particular importance to analyse all OCT scans of each examination of eyes with lamellar macular hole. Based on high resolution OCT analysis, future studies may help to define subgroups of lamellar macular hole that are eligible for macular surgery by differentiation of lamellar hole-associated epiretinal proliferation from contractive epiretinal membranes.
ACKNOWLEDGMENT ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST. The authors indicate no financial conflict of interest involved in design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, and approval of the manuscript. This study was supported by unrestricted grants of the Ludwig-Maximilians-University, Munich, Germany, Grant for Research and Education (FöFoLe, ID # 822). Involved in design and conduct of study (D.C., R.G.S.); collection, management, analysis and interpretation of data (D.C., C.H., A.W., R.G.S.); specimen preparation (D.C., E.E., R.S.) and critical review and approval (D.C., C.H., W.J.M., A.W., A.K., R.G.S.) of manuscript. [10]
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REFERENCES 1. Haouchine B, Massin P, Tadayoni R, Erginay A, Gaudric A. Diagnosis of macular pseudoholes and lamellar macular holes by optical coherence tomography. Am J Ophthalmol 2004;138(5):732-739.
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2. Witkin AJ, Ko TH, Fujimoto JG, et al. Redefining lamellar holes and the vitreomacular interface: an ultrahigh-resolution optical coherence tomography study. Ophthalmology 2006;113(3):388-397.
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3. Parolini B, Schumann RG, Cereda MG, Haritoglou C, Pertile G. Lamellar macular hole: a clinicopathologic correlation of surgically excised epiretinal membranes. Invest Ophthalmol Vis Sci 2011;52(12):9074-9083.
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4. Michalewska Z, Michalewski J, Odrobina D, Nawrocki J. Non-full-thickness macular holes reassessed with spectral domain optical coherence tomography. Retina 2012;32(5):922-929. 5. Bottoni F, Deiro AP, Giani A, Orini C, Cigada M, Staurenghi G. The natural history of lamellar macular holes: a spectral domain optical coherence tomography study. Graefes Arch Clin Exp Ophthalmol 2013;251(2):467-475.
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6. Schumann RG, Compera D, Schaumberger MM, et al. Epiretinal membrane characteristics correlate with photoreceptor layer defects in lamellar macular holes and macular pseudoholes. Retina 2015;35(4):727-735. 7. Pang CE, Spaide RF, Freund KB. Epiretinal proliferation seen in association with lamellar macular holes: a distinct clinical entity. Retina 2014;34(8):1513-1523.
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8. Pang CE, Spaide RF, Freund KB. Comparing functional and morphologic characteristics of lamellar macular holes with and without lamellar hole-associated epiretinal proliferation. Retina 2015;35(4):720-726.
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9. Duker JS, Kaiser PK, Binder S, et al. The International Vitreomacular Traction Study Group classification of vitreomacular adhesion, traction, and macular hole. Ophthalmology 2013;120(12):2611-2619. 10. Gass JD. Lamellar macular hole: a complication of cystoid macular edema after cataract extraction: a clinicopathologic case report. Trans Am Ophthalmol Soc 1975;73:231-250. 11. Theodossiadis PG, Grigoropoulos VG, Emfietzoglou I, et al. Evolution of lamellar macular hole studied by optical coherence tomography. Graefes Arch Clin Exp Ophthalmol 2009;247(1):13-20. 12. Androudi S, Stangos A, Brazitikos PD. Lamellar macular holes: tomographic features and surgical outcome. Am J Ophthalmol 2009;148(3):420-426. [11]
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13. Zhao F, Gandorfer A, Haritoglou C, et al. Epiretinal cell proliferation in macular pucker and vitreomacular traction syndrome: analysis of flat-mounted internal limiting membrane specimens. Retina 2013;33(1):77-88.
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14. Schumann RG, Schaumberger MM, Rohleder M, Haritoglou C, Kampik A, Gandorfer A. Ultrastructure of the vitreomacular interface in full-thickness idiopathic macular holes: a consecutive analysis of 100 cases. Am J Ophthalmol 2006;141(6):1112-1119.
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15. Schumann RG, Eibl KH, Zhao F, et al. Immunocytochemical and ultrastructural evidence of glial cells and hyalocytes in internal limiting membrane specimens of idiopathic macular holes. Invest Ophthalmol Vis Sci 2011;52(11):7822-7834.
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16. Arora PD, McCulloch CA. Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts. J Cell Physiol 1994;159(1):161-175. 17. Hinz B. It has to be the αv: myofibroblast integrins activate latent TGF-beta1. Nat Med 2013;19(12):1567-1568. 18. Bringmann A, Wiedemann P. Involvement of Muller glial cells in epiretinal membrane formation. Graefes Arch Clin Exp Ophthalmol 2009;247(7):865-883.
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19. Dingemans KP, Teeling P. Long-spacing collagen and proteoglycans in pathologic tissues. Ultrastruct Pathol 1994;18(6):539-547.
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20. Ishida S, Yamazaki K, Shinoda K, Kawashima S, Oguchi Y. Macular hole retinal detachment in highly myopic eyes: ultrastructure of surgically removed epiretinal membrane and clinicopathologic correlation. Retina 2000;20(2):176-183.
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FIGURE LEGENDS
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Figure 1. Lamellar hole-associated epiretinal proliferation. (Top left, Top right) Spectraldomain optical coherence tomography images of a 70-years old female presenting lamellar macular hole with lamellar hole-associated epiretinal proliferation (white arrowhead). (Middle left) Immunolabelling and cell nuclei staining (blue) of flat-mounted specimen demonstrate positive immunoreactivity for the hyalocyte cell marker antiCD64 (red) and (Middle right) positive immunoreactivity for the glial cell marker anti-glial fibrillary acidic protein (green). (Bottom left) Transmission electron micrograph of lamellar hole-associated epiretinal proliferation shows abundant cell agglomeration of fibroblasts and hyalocytes. (Bottom right) Rectangle of (Bottom left) in detail, representing fibroblasts characterized by abundant rough endoplasmatic reticulum (black arrow). (Original magnification: (C) x100; (D) x400; (E) x3,000; (F) x120,000)
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Figure 2. Combination of both lamellar hole-associated epiretinal proliferation and conventional epiretinal membrane. (Top left, Top right) Spectral-domain optical coherence tomography images of a 79-years old male with lamellar macular hole associated with both a conventional epiretinal membrane (white arrow) and lamellar hole-associated epiretinal proliferation (white arrowhead). (Middle left) Light micrograph with toluidine blue staining of serial semi-thin sections shows a compact and tightly stretched epiretinal membrane on folded internal limiting membrane. (Middle right) Light micrograph demonstrates abundant epiretinal cell proliferation embedded in extracellular matrix of a specimen with lamellar hole-associated epiretinal proliferation. (Bottom left) Transmission electron micrograph shows myofibroblasts of conventional epiretinal membrane located on folded vitreous collagen strand (NVC) (black arrow). (Bottom right) Lamellar hole-associated epiretinal proliferation was seen as compact cell agglomeration with abundant fibroblasts. (Original magnification: (C, D) x200; (E) x1,800, (F) x1,800)
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Figure 3. Conventional epiretinal membrane of macular pseudohole. (Top left, Top right) Spectral-domain optical coherence tomography images of a 72-years old male with macular pseudohole and conventional epiretinal membrane (white arrow). (Middle left) Immunolabelling and cell nuclei staining (blue) of flat-mounted specimen demonstrate positive immunoreactivity for the myofibroblast cell marker anti-α-smooth muscle actin (red) and (Middle right) positive immunoreactivity for the glial cell marker anti-glial fibrillary acidic protein (green). (Bottom left) Transmission electron micrograph of conventional epiretinal membrane shows cell multilayer with myofibroblasts located on native vitreous collagen on the vitreal side of the internal limiting membrane (asterisk). (Bottom right) Transmission electron micrograph representing myofibroblast located on a thick vitreous cortex collagen strand with embedded fibrous long spacing collagen (detail). (Original magnification: (C, D) x100; (E) x3,000; (F) x4,400)
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Meta-
Sex/Age
Diagnosis
morphopsia
Preop BCVA
Postop BCVA
Preop state
Postop state
Follow-up
(+/-)
(LogMAR)
(LogMAR)
of the lens
of the lens
(months)
IOL
21
IOL
18
M/74
LMH
-
0.5
0.1
Phakic
2
M/71
LMH
+
0.3
0.4
Phakic
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
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1
SC
No
RI PT
Table 1. Clinical data of patients with lamellar macular hole and macular pseudohole.
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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
23
F/64
MPH
-
0.2
0.2
Phakic
24
M/77
MPH
-
0.4
0.4
Phakic
25
F/72
MPH
-
0.2
0.2
Phakic
SC
RI PT
20
IOL
4
IOL
3
IOL
4
IOL
3
AC C
EP
TE D
M AN U
M, male; F, female; LMH, lamellar macular hole; MPH, macular pseudohole; BCVA, bestcorrected visual acuity; IOL, intraocular lens.
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Table 2. Spectral-domain optical coherence tomography analysis and cell distribution analysis of specimens removed from eyes with lamellar macular holes and macular pseudoholes. SD-OCT analysis
Area of
peeled
State of posterior hyaloid
ERM
No of
cells
ILM
LHEP
2
(mm ) LMH
complete PVD
x
-
6.9
2246
2
LMH
complete PVD
x
x
13.32
4942
3
LMH
complete PVD
x
-
15.44
4
LMH
complete PVD
x
x
5
LMH
partial PVD
-
x
6
LMH
complete PVD
x
x
7
LMH
complete PVD
x
-
8
LMH
partial PVD
-
9
LMH
complete PVD
10
LMH
complete PVD
11
LMH
complete PVD
(cells/mm )
Single cells
Homogenous
Cluster of cells
layer of cells
326
-
x
-
371
-
x
-
2651
172
-
x
-
3.62
2583
714
-
x
-
6.74
779
116
-
x
-
7.06
867
123
-
x
-
1.63
405
248
-
x
-
1.93
175
91
-
-
x
TE D
EP
AC C
x
Cell distribution
2
M AN U
1
Cell density
RI PT
Diagnosis
SC
No
-
x
2.27
322
142
-
-
x
x
x
1.81
995
550
-
x
-
x
x
2.24
898
401
-
x
-
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MPH
complete PVD
x
-
34.86
17771
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
25
MPH
complete PVD
EP
TE D
M AN U
SC
RI PT
12
AC C
-
x
-
4.09
889
217
x
-
-
x
-
4.2
1669
397
-
x
-
LMH, lamellar macular hole; MPH, macular pseudohole; PVD, posterior vitreous detachment; SD-OCT, Spectral-domain optical coherence tomography; ERM, epiretinal membrane; LHEP, lamellar hole-associated epiretinal proliferation; ILM, internal limiting membrane.
ACCEPTED MANUSCRIPT Table 3. Immunoreactivity in flat-mounted specimens removed from eyes with lamellar macular hole and macular pseudohole.
Anti-
Target structure
Immunoreactivity LMH
MPH
(N = 11)
(N = 14)
++
+
Intermediate type
(GFAP)
filaments of glial cells
Vimentin
Mueller cells, astrocytes
++
(+)
Alpha- smooth muscle actin (α-
Intracellular actin
(+)
++
SMA)
filaments
CD 45
Hyalocytes
++
+
CD 64
Hyalocytes
++
+
Collagen type I
Newly formed collagen
++
+
Collagen type II
Vitreous cortex collagen
+
(+)
Collagen type III
Extracelluar matrix
++
(+)
Laminin
Basalmembrane-ILM
++
++
M AN U
SC
RI PT
Glial fibrillary acidic protein
LMH, lamellar macular hole; MPH, macular pseudohole; ILM, internal limiting membrane.
AC C
EP
TE D
++, very present; +, present; (+), sparsely present; -, absent.
ACCEPTED MANUSCRIPT Table 4. Ultrastructural features of specimens removed from eyes with lamellar macular hole and macular pseudohole. specimens (N = 25)
Ultrastructural features
LMH (N = 11 ) 9
MPH (N = 14 ) 14
Single cells
0
1
Monolayer of cells
1
1
Multilayer of cells
3
12
Cell agglomeration
7
0
Hyalocytes
6
8
Macrophages
0
Fibrous astrocytes
1
Myofibroblasts
4
Inner limiting membrane
1 1
12
M AN U
Fibroblasts
SC
Cell types
RI PT
Cell distribution at the ILM
8
3
0
1
1
7
4
12
6
4
Native vitreous collagen
9
14
Newly formed collagen
6
7
Fibrous long spacing collagen
3
7
Retinal pigment epithelial cells Collagen distribution Thick vitreous collagen strands Vitreous collagen on ILM Collagen dispersed with cells
TE D
Collagen types
AC C
EP
LMH, lamellar macular hole; MPH, macular pseudohole; ILM, inner limiting membrane.
AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
SC
RI PT
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AC C
EP
TE D
M AN U
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Denise Compera, MD, is currently undergoing her residency at the Department of Ophthalmology, Ludwig-Maximilians-University Munich, Germany. Since 2012, she joined the research group of “Vitreoretinal pathology and electron microscopy“. Dr Compera’s research interests include diseases of the vitreoretinal interface, in particular imaging techniques and surgical intervention in traction maculopathies.
AC C
EP
TE D
M AN U
SC
RI PT
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