Ultrastructural Details of Epiretinal Membrane Foveoschisis.

INTRODUCTION: The aim of this study was to describe differences in the vitreomacular interface (VMI) in idiopathic epiretinal membrane (ERM) foveoschisis compared to macular pseudohole (MPH) and lamellar macular hole (LMH). METHODS: We analysed surgically excised epiretinal material and internal limiting membrane (ILM) specimens obtained from 16 eyes of 16 patients with ERM foveoschisis (6 eyes), MPH (5 eyes), and LMH (5 eyes) during standard pars plana vitrectomy (PPV) with membrane peeling. The three entities were classified according to the newly introduced optical coherence tomography (OCT) terminology. Transmission electron microscopy (TEM) was used to describe the ultrastructural features. RESULTS: We found fibrocellular epiretinal tissues in all samples analysed. However, the cell and collagen composition of the VMI differed between groups. Eyes with ERM foveoschisis were characterized by a higher number of cells, multilayered membranes, and thick strands of vitreous collagen embedding the major cell types of myofibroblasts compared to MPH. Eyes with MPH also showed a predominance of myofibroblasts, but these were located directly on the ILM with no collagen between the cells and the ILM. Eyes with LMH showed a thick, multilayered epiretinal proliferation consisting mainly of non-tractional glial cells, corresponding to hypodense epiretinal proliferation on OCT. Eyes with ERM foveoschisis and MPH were more likely to have incomplete PVD compared to LMH in terms of posterior hyaloid status. DISCUSSION/CONCLUSION: Tractional ERMs in eyes with ERM foveoschisis and MPH differ in their ultrastructure. The main difference is in the amount and topographical distribution of vitreous collagen. However, the epiretinal cell types are predominantly myofibroblasts in both entities. This highlights the importance of distinguishing ERM foveoschisis from both MPH and LMH in terms of pathogenesis and surgical peeling procedures.

PORES OF THE INTERNAL LIMITING MEMBRANE: A Common Finding in Vitreomaculopathies.

PURPOSE: To describe presence and distribution of pores of the inner limiting membrane (ILM) in eyes with vitreomaculopathies. METHODS: Inner limiting membrane specimens were harvested from 117 eyes of 117 patients during vitrectomy with membrane peeling from eyes with vitreomacular traction syndrome, idiopathic and secondary epiretinal gliosis, and idiopathic full-thickness macular hole. All specimens were processed as flat-mounts for immunocytochemistry and examined by phase-contrast, interference, and fluorescence microscopy. Demographic and clinical data were correlated. RESULTS: Inner limiting membrane pores were found in all vitreomaculopathies. They were identified in 47 (40.2%) of 117 eyes being most evident with antilaminin. In eyes with full-thickness macular hole >400 µ m, pores were seen in more than half of all eyes. They occur as numerous and uniformly distributed defects of the flat-mounted ILM with a mean diameter of 9.5 ± 2.4 µ m. Edges of ILM pores are round with an irregular contour and no specific cellular pattern. Pores were distinguished from retinal vessel thinning and iatrogenic artefacts. CONCLUSION: Contrary to previous reports, ILM pores are a common finding in vitreomaculopathies easily visible with antilaminin staining. Further studies are needed to clarify whether their presence correlates with differences in disease progression or imaging before and after vitrectomy with ILM peeling.

Cell composition at the vitreomacular interface in traumatic macular holes.

PURPOSE: To describe characteristics of the vitreomacular interface (VMI) in traumatic macular holes (TMH) compared to idiopathic macular holes (IMH) using immunofluorescence and electron microscopy, and to correlate with clinical data. METHODS: For immunocytochemical and ultrastructural analyses, premacular tissue with internal limiting membrane (ILM) and epiretinal membrane (ERM) was harvested during vitrectomy from 5 eyes with TMH and 5 eyes with IMH. All specimens were processed as flat mounts for phase-contrast microscopy, interference and fluorescence microscopy, and transmission electron microscopy (TEM). Primary antibodies were used against microglial and macroglial cells. Clinical data was retrospectively evaluated. RESULTS: Surgically excised premacular tissue of eyes with TMH showed a less pronounced positive immunoreactivity for anti-glutamine synthetase, anti-vimentin and anti-IBA1 compared to eyes with IMH. Cell nuclei staining of the flat-mounted specimens as well as TEM presented a lower cell count in eyes with TMH compared to IMH. All detected cells were found on the vitreal side of the ILM. No collagen fibrils were seen in specimens of TMH. According to patients' age, intraoperative data as well as spectral-domain optical coherence tomography (SD-OCT) analysis revealed an attached posterior vitreous in the majority of TMH cases (60%), whereas all eyes with IMH presented posterior vitreous detachment. CONCLUSION: The vitreomacular interface in TMH and IMH shows significant differences. In TMH, glial cells are a rare finding on the vitreal side of the ILM.

Foveal Abnormality associated with epiretinal Tissue of medium reflectivity and Increased blue-light fundus Autofluorescence Signal (FATIAS).

PURPOSE: To describe a distinct vitreomacular interface disorder (VMID) termed Foveal Abnormality associated with epiretinal Tissue of medium reflectivity and Increased blue-light fundus Autofluorescence Signal (FATIAS). METHODS: A case series including forty-seven eyes of 47 patients. The included eyes must present an irregular foveal contour on optical coherence tomography (OCT) and a pathologically increased autofluorescent signal at the fovea on blue-light fundus autofluorescence (B-FAF). Main outcome measures were morphologic characteristics of the lesions, logarithm of minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA), and central foveal thickness (CFT). RESULTS: The following two types of FATIAS were identified: (1) the step type characterized by an asymmetric contour of the foveal pit and by a tissue of medium reflectivity on the foveal surface and (2) the rail type characterized by a shallow foveal pit and a rail of tissue of medium reflectivity on the foveal surface. The outer retinal bands were continuous in all cases. Both types presented with an area of increased B-FAF signal, usually bilobed in the step type and round and centered on the foveal pit in the rail type. LogMAR BCVA was 0.09 ± 0.1 and 0.1 ± 0.1 (P = 0.91), and CFT was 197.8 ± 9.7 and 202.2 ± 13.2 (P = 0.19) in the step and in the rail group, respectively. CONCLUSIONS: We describe a distinct VMID named FATIAS. Two types of FATIAS may be appreciated with SD-OCT and B-FAF analyses, the step and the rail type. Both are characterized by abnormal foveal contour and autofluorescence signal.

Premacular Cell Proliferation Profiles in Tangential Traction Vitreo-Maculopathies Suggest a Key Role for Hyalocytes.

PURPOSE: To compare immunocytochemical and ultrastructural features of premacular tissue surgically removed from eyes with tangential traction vitreo-maculopathies. METHODS: By spectral-domain optical coherence tomography (SD-OCT), premacular tissue was differentiated into premacular proliferation and premacular membrane (PMM). Specimens were harvested during vitrectomy from 10 eyes with macular pucker, lamellar macular hole (LMH) and full-thickness macular hole, and prepared for immunocytochemistry and transmission electron microscopy. RESULTS: All specimens showed positive autofluorescence consistent with the yellow colour of peeled tissue. Glial cells were predominantly positive in premacular proliferation. Hyalocytes were the main cell type in PMM. Electron microscopy revealed densely packed premacular glial cells neighbouring hyalocytes and vitreous collagen strands. Myofibroblasts with features indicative of contractile properties were found in PMM, exclusively. Cell composition of premacular proliferation was free of contractile elements. CONCLUSION: All three types of vitreo-maculopathy have similar cell constituents in their premacular tissue. Cell population of premacular proliferation is not unique for LMHs. Corresponding to SD-OCT, electron microscopy demonstrates hyalocytes and vitreous collagen in PMMs both directly adjacent to the cellular complex of premacular proliferation. Study results point to the vitreous as one important pathogenic player potentially driving the degenerative cellular process at the vitreoretinal interface in tangential traction vitreo-maculopathies.

Premacular membranes in tissue culture.

PURPOSE: To investigate integrity and characteristics of human premacular membranes (PMM) with and without standard tissue culturing using mechanical traction. METHODS: Premacular membranes were harvested from 32 eyes of 32 patients with idiopathic macular pucker during standard vitrectomy. By flat-mount preparation with phase contrast and interference microscopy, specimens were prepared for time-lapse microscopy, immunocytochemistry, and transmission electron microscopy. Sixteen of 32 specimens were held in tissue culture with tangential traction by using entomological pins. Of these, specimens of 7 eyes were analyzed with and without tissue culturing for comparison. Primary antibodies were used for myofibroblasts, hyalocytes, macro-/microglial cells, and retinal pigment epithelial and immune cells. RESULTS: Hyalocytes, macroglia, and microglia composed the main cell composition of surgically removed PMM. Correlation of time-lapse microscopy with immunofluorescence microscopy identified fast and unidirectional moving small round cells as microglia. Slowly moving elongated large cells were characterized as alpha-smooth muscle actin (α-SMA)-positive myofibroblasts. Following tissue culturing with tangential stretch, enhanced positive immunolabelling for α-SMA and integrins-αv was seen. All other labelling results were demonstrated to be similar with pre-culture conditions. Ultrastructural analysis revealed fibroblasts, myofibroblasts, and proliferation of glial cells following tissue culture. CONCLUSION: This study demonstrates abundance of fibroblasts, myofibroblasts, and glial cells in PMM from idiopathic macular pucker following tissue culture with tangential stretch application. We found enhanced contractive properties of the cultured PPM that appear to indicate transdifferentiation of the cell composition. This in vitro model may improve understanding of pathogenesis in traction maculopathies and help to establish further anti-fibrosis treatment strategies.

VITRECTOMY FOR INTERMEDIATE AGE-RELATED MACULAR DEGENERATION ASSOCIATED WITH TANGENTIAL VITREOMACULAR TRACTION: A CLINICOPATHOLOGIC CORRELATION.

PURPOSE: To describe the morphologic characteristics of the vitreomacular interface in intermediate age-related macular degeneration associated with tangential traction due to premacular membrane formation and to correlate with optical coherence tomography (OCT) findings and clinical data. METHODS: Premacular membrane specimens were removed sequentially with the internal limiting membrane from 27 eyes of 26 patients with intermediate age-related macular degeneration during standard vitrectomy. Specimens were processed for immunocytochemical staining of epiretinal cells and extracellular matrix components. Ultrastructural analysis was performed using transmission electron microscopy. Spectral domain optical coherence tomography images and patient charts were evaluated in retrospect. RESULTS: Immunocytochemistry revealed hyalocytes and myofibroblasts as predominant cell types. Ultrastructural analysis demonstrated evidence of vitreoschisis in all eyes. Myofibroblasts with contractile properties were observed to span between folds of the internal limiting membrane and vitreous cortex collagen. Retinal pigment epithelial cells or inflammatory cells were not detected. Mean visual acuity (Snellen) showed significant improvement from 20/72 ± 20/36 to 20/41 ± 20/32 (P < 0.001) after a mean follow-up period of 19 months (median, 17 months). During this period, none of the eyes required anti-vascular endothelial growth factor therapy. CONCLUSION: Fibrocellular premacular proliferation in intermediate age-related macular degeneration predominantly consists of vitreous collagen, hyalocytes, and myofibroblasts with contractile properties. Vitreoschisis and vitreous-derived cells appear to play an important role in traction formation of this subgroup of eyes. In patients with intermediate age-related macular degeneration and contractile premacular membrane, release of traction by vitrectomy with internal limiting membrane peeling results in significantly functional and anatomical improvement.

COMPLIANCE AND ADHERENCE OF PATIENTS WITH DIABETIC MACULAR EDEMA TO INTRAVITREAL ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY IN DAILY PRACTICE.

PURPOSE: We assessed differences in compliance and adherence (lateness of patients, visual acuity, reasons for abstaining) between patients with diabetic macular edema (DME) and patients with age-related macular degeneration (AMD), both under anti-vascular endothelial growth factor therapy. METHODS: We included 136 patients with DME (36% women, 65 years, 22 visits, 13.9 injections, and 29.9 months of follow-up) and 109 patients with AMD (59% women, 76 years, 20 visits, 14.7 injections, and 22.3 months of follow-up) (minimum follow-up of 12 months and at least 5 injections). We assessed missed appointments (lateness >14 days) and therapy break-offs (lateness >100 days). All delayed patients were called and interviewed for abstaining reasons. RESULTS: Forty-six percent of patients with DME and 22% of patients with AMD had at least one break-off. Thirty-five percent of patients with DME and 50% of patients with AMD were always on schedule. In patients with DME, there was significant correlation (P = 0.017) between the number of break-offs and change of visual acuity. In 60% DME and 38% AMD of break-off cases, visual acuity was worse than the before break-off. The most common reason for abstaining was comorbidities (33% AMD and 20% DME). CONCLUSION: There are significant differences between patients with AMD and DME regarding compliance and adherence, which also affects outcome. Strategies to tie patients with DME to costly intravitreal therapy need to be developed to improve outcomes and efficacy.

Impact of Preinjection Spectral Domain Optical Coherence Tomography Findings in the Use of Intravitreal Ocriplasmin in a Clinical Setting.

PURPOSE: To investigate the impact of spectral domain optical coherence tomography (SD-OCT) morphological predictive markers on visual acuity and outcome using ocriplasmin for macular hole and vitreomacular traction syndrome. METHODS: A series of 40 patients in a retrospective study received intravitreal ocriplasmin. The primary endpoint was defined as morphological resolution of vitreomacular traction or closure of a macular hole. We analyzed the impact of pre- and postinjection SD-OCT findings on the outcome and visual acuity. RESULTS: Thirteen of the 40 patients benefited from treatment. Statistical correlation between baseline characteristics and outcome revealed that higher foveal thickness (p = 0.018) and nontractional epiretinal membranes (p = 0.05) resulted in a worse outcome. In treatment success best corrected visual acuity gained was 9 (SD 12) letters and in failure 1 (SD 9) letter. We could not observe an influence of preinjection SD-OCT findings and other factors on visual outcome. CONCLUSION: We could confirm the therapeutic effect of ocriplasmin injections. SD-OCT morphological factors that influence treatment success and visual acuity were determined.

In Vitro Comparison of 2D-Cell Culture and 3D-Cell Sheets of Scleraxis-Programmed Bone Marrow Derived Mesenchymal Stem Cells to Primary Tendon Stem/Progenitor Cells for Tendon Repair.

The poor and slow healing capacity of tendons requires novel strategies to speed up the tendon repair process. Hence, new and promising developments in tendon tissue engineering have become increasingly relevant. Previously, we have established a tendon progenitor cell line via ectopic expression of the tendon-related basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) in human bone marrow mesenchymal stem cells (hMSC-Scx). The aim of this study was to directly compare the characteristics of hMSC-Scx cells to that of primary human tendon stem/progenitors cells (hTSPCs) via assessment of self-renewal and multipotency, gene marker expression profiling, in vitro wound healing assay and three-dimensional cell sheet formation. As expected, hTSPCs were more naive than hMSC-Scx cells because of higher clonogenicity, trilineage differentiation potential, and expression of stem cell markers, as well as higher mRNA levels of several gene factors associated with early tendon development. Interestingly, with regards to wound healing, both cell types demonstrate a comparable speed of scratch closure, as well as migratory velocity and distance in various migration experiments. In the three-dimensional cell sheet model, hMSC-Scx cells and hTSPCs form compact tendinous sheets as histological staining, and transmission electron microscopy shows spindle-shaped cells and collagen type I fibrils with similar average diameter size and distribution. Taken together, hTSPCs exceed hMSC-Scx cells in several characteristics, namely clonogenicity, multipotentiality, gene expression profile and rates of tendon-like sheet formation, whilst in three-dimensional cell sheets, both cell types have comparable in vitro healing potential and collagenous composition of their three-dimensional cell sheets, making both cell types a suitable cell source for tendon tissue engineering and healing.

The predictability of ocriplasmin treatment effects: is there consensus among retinal experts? Results from the EXPORT study.

PURPOSE: To evaluate the agreement and predictability of ocriplasmin treatment effects among retinal experts (raters) by assessment of retinal imaging data of eyes treated for vitreomacular traction in nine different centers in Germany and Austria. METHODS: Retrospective cohort study. Combined confocal near-infrared scanning laser ophthalmoscopy and spectral-domain optical coherence tomography images (Spectralis® device, Heidelberg Engineering GmbH, Germany) from 136 eyes of 135 subjects were reviewed by 14 raters using an internet-based grading database and a standardized questionnaire. In addition to the images taken within 2 days prior to treatment, age, gender, and lens status were disclosed to the raters. Treatment success was defined as a complete cleavage of the posterior vitreous cortex at day 28±5. Main outcome was the agreement and predictability among raters for assessment of treatment success. RESULTS: Raters generally accepted starting ocriplasmin treatment (chance for treatment success ≥ 1%) in 22.4 to 69.1% (median 53.2%) of eyes (moderate intra- and interrater agreements with kappa-values of 0.6 and 0.48). The likelihood for a high potential treatment success (equal or higher than 25%) was judged by the raters in 43.4% to 86.0% (median 62.6%) of eyes (moderate intra- and fair interrater agreements with kappa-values of 0.56 and 0.22). Allocating eyes for high potential treatment success overall increased the odds by 3.07, with odds ratios of single raters up to 4.06 to 6.16. CONCLUSIONS: These results underscore the importance of training health care providers in the evaluation of retinal imaging data and also to define characteristic morphological features better in the presence of vitreoretinal interface diseases. The better results of single raters in the predictability of treatment success by the allocation of eyes in the high-potential group indicates the high relevance of the meticulous analysis of retinal images.

Assessment of intravitreal ocriplasmin treatment for vitreomacular traction in clinical practice.

PURPOSE: To assess treatment effects following intravitreal injection of ocriplasmin for vitreomacular traction (VMT), with or without full-thickness macular hole (FTMH), in real-life setting. METHODS: This is a monocentric, retrospective, consecutive series of 82 eyes from 82 patients who underwent ocriplasmin treatment between July 2013 and December 2016. We included 57 eyes with pure VMT, 17 eyes with small FTMHs, and eight eyes with medium FTMHs. Primary outcome measures were VMT release and MH closure rates. Secondary outcomes were visual acuity (VA), morphological changes, and subjective visual impairment after 1, 3, and 6 months and at last follow-up. RESULTS: After a median follow-up of 10 months, VMT release was achieved by pharmacologic vitreolysis in 57% of all eyes, whereas the macular hole closure rate was 32%. In those presenting with five or more positive prognostic factors (PPF), eyes with pure VMT showed nonsurgical traction release in 88%, and FTMHs were released in 93%, with a closure rate of 20%. Small FTMHs closed in 41% and medium FTMHs in 13%. The mean change in VA (LogMAR) was -0.07 ± 0.24 (median - 0.10) in all eyes. Subretinal fluid accumulation and ellipsoid zone changes were seen in 31% and 37% of all eyes, respectively. They were more frequent in eyes with traction release, but were self-limited. CONCLUSIONS: In a real-life setting, release of VMT by ocriplasmin injection can be achieved in the majority of eyes, relying on a strict patient selection. Closure of FTMHs rather correlates with hole diameter than with presence of PPF, and remains a rare finding in medium FTMHs.

A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

PURPOSE: To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. METHODS: Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. RESULTS: Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. CONCLUSIONS: This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

VITRECTOMY FOR PERSISTENT MACULAR HOLES FOLLOWING OCRIPLASMIN INJECTION: A Comparative Multicenter Study.

PURPOSE: To determine functional and anatomical outcomes of pars plana vitrectomy for persistent full-thickness macular hole (MH) after intravitreal injection of ocriplasmin. METHODS: This is a multicenter retrospective interventional study of 37 eyes of 37 patients who underwent pars plana vitrectomy with internal limiting membrane peeling for persistent MH after ocriplasmin treatment between December 2013 and December 2015 and comparison with 35 eyes of 35 patients who were offered ocriplasmin injection but underwent pars plana vitrectomy alone without pharmacologic vitreolysis before surgery. In addition, 24 matched pairs (MH diameter at baseline ±5 µm) were analyzed. Clinical data such as visual acuity, intraoperative characteristics, and spectral domain optical coherence tomography images were reviewed. Main outcome measures were visual acuity and MH closure rate. RESULTS: After a mean follow-up period of 9 months, postoperative mean visual acuity showed no significant differences between ocriplasmin-treated eyes (logarithm of minimum angle of resolution 0.37 ± 0.26, Snellen 20/47) and eyes without ocriplasmin treatment (logarithm of minimum angle of resolution 0.39 ± 0.25; Snellen 20/49) (P > 0.9). After ocriplasmin injection, mean MH diameter enlarged from 217 ± 102 µm to 384 ± 239 µm (P < 0.001). Matched-pair analysis revealed no difference in gain of visual acuity between the first visit and the last follow-up (P = 0.29). Macular hole closure was observed in similar proportion in ocriplasmin-treated eyes (97%) and vitrectomy-only eyes (94%) (P > 0.5). CONLCUSION: Eyes with persistent MH after ocriplasmin injection showed significant visual improvement after pars plana vitrectomy. Matched-pair analysis revealed no statistical differences in functional and anatomical postoperative results comparing with eyes of similar MH diameter that proceeded directly to surgery without ocriplasmin pretreatment.

Propiedades biomecánicas de la membrana limitante interna tras recibir tratamiento intravítreo con ocriplasmina.

Objetivo: Evaluar la rigidez de la membrana limitante interna (MLI) humana y evaluar los posibles cambios de las propiedades mecánicas tras administrar una inyección intravítrea de ocriplasmina para tratar la tracción vitreomacular. Métodos: Este estudio se compone de una serie de casos intervencionales y comparativos de 12 muestras de MLI extraídas mediante cirugía y obtenidas de forma consecutiva de 9 ojos de 9 pacientes después de someterse sin éxito a vitreólisis farmacológica con ocriplasmina. Durante el mismo periodo de tiempo, 16 muestras de otros 13 ojos sin tratamiento con ocriplasmina se obtuvieron mediante vitrectomía y sirvieron como controles. Todos los pacientes presentaron agujeros maculares o tracción vitreomacular y se sometieron a vitrectomía con disección de la MLI tanto con tinción con azul brillante (AB) como sin ella. Todas las muestras se analizaron con un microscopio de fuerza atómica con imágenes de las regiones de 25 × 25 µm. En todas las muestras, se analizaron tanto la parte de la retina como la del vítreo de la MLI. Resultados: La microscopia de fuerza atómica no reveló diferencias significativas en cuanto a elasticidad de las muestras de MLI extraídas de ojos con o sin tratamiento con ocriplasmina. Las áreas onduladas de la parte de la retina presentaron una mayor rigidez que la parte del vítreo de la MLI. La cartografía topográfica tanto de la parte del vítreo como de la retina de la MLI no mostró ninguna alteración aparente de la morfología en ojos tratados con ocriplasmina en comparación con los ojos no tratados. La tinción con azul brillante conllevó un aumento de la rigidez tisular. Conclusiones: Las inyecciones intravítreas de ocriplasmina no varían las propiedades biomecánicas de la MLI humana. No existen pruebas de un posible efecto enzimático que interfiera con la rigidez de esta membrana basal.

Biomechanical Properties of the Internal Limiting Membrane after Intravitreal Ocriplasmin Treatment.

PURPOSE: To assess the stiffness of the human internal limiting membrane (ILM) and evaluate potential changes of mechanical properties following intravitreal ocriplasmin injection for vitreomacular traction. METHODS: This is an interventional comparative case series of 12 surgically excised ILM specimens consecutively obtained from 9 eyes of 9 patients after unsuccessful pharmacologic vitreolysis with ocriplasmin. During the same time period, 16 specimens from 13 other eyes without ocriplasmin treatment were harvested during vitrectomy and served as controls. All patients presented with macular holes or vitreomacular traction and underwent vitrectomy with ILM peeling either with or without brilliant blue (BB) staining. All specimens were analyzed using atomic force microscopy with scan regions of 25 × 25 µm. In all specimens, both the retinal side and vitreal side of the ILM were analyzed. RESULTS: Atomic force microscopy revealed no significant differences in elasticity of ILM specimens removed from eyes with or without ocriplasmin treatment. Undulated areas of the retinal side presented stiffer than the vitreal side of the ILM. Topographical mapping of both the vitreal and retinal side of the ILM showed no apparent alteration of the morphology in ocriplasmin-treated eyes compared to untreated eyes. Staining with BB resulted in an increase of tissue stiffness. CONCLUSIONS: Intravitreal injection of ocriplasmin does not change biomechanical properties of the human ILM. There is no evidence of a potential enzymatic effect of ocriplasmin interfering with the stiffness of this basement membrane.

Cells at the vitreoretinal interface in small full-thickness macular holes.

PURPOSE: To report on total number, distribution, and type of cells at the vitreomacular interface in small full-thickness macular holes. METHODS: Internal limiting membrane specimens were removed from 20 consecutive patients with macular holes <250 µm at times when pharmacologic vitreolysis was not available. Specimens were flat mounted and investigated by phase contrast and interference microscopy and immunocytochemistry. Clinical data were documented including optical coherence tomography analysis using the caliper function. Thirteen antibodies were used for glial cells, hyalocytes, macrophages, retinal pigment epithelial cells, different types of collagen, alpha-smooth muscle actin, and proliferating cells. RESULTS: There was a positive correlation between macular hole size and cell density at the internal limiting membrane (Spearman's Rho: r = 0.519, P = 0.019). Mostly, single glial cells were found on the internal limiting membrane. In five patients, cell clusters were present. There was a strong immunoreactivity for glial cell markers. Immunoreactivity of hyalocyte markers, alpha-smooth muscle actin, and Ki-67 was found in cell clusters but otherwise sparse. CONCLUSION: Single cells of glial origin without signs of proliferation or contraction are present in eyes with small full-thickness macular holes. In some eyes, however, clusters of cells can be seen, capable of proliferation and exerting tangential traction. Our findings emphasize the need for better visualization of the vitreoretinal pathology by optical coherence tomography, especially to distinguish between single cells and cell clusters.

Epiretinal membrane characteristics correlate with photoreceptor layer defects in lamellar macular holes and macular pseudoholes.

PURPOSE: To report on epiretinal membrane (ERM) characteristics and photoreceptor layer integrity of lamellar macular holes (LMHs) and macular pseudoholes (MPHs), and to compare with clinical course in operated and untreated eyes. METHODS: We consecutively reviewed the charts of patients with LMH and MPH between 2003 and 2013. For clinical analysis, we included 87 eyes (48 with LMH, 39 with MPH) with a minimum follow-up of 6 months. Of these, we included 64 eyes (37 with LMH, 27 with MPH) for high-resolution spectral domain optical coherence tomography analysis with examinations fulfilling the required resolution and quality of optical coherence tomography images. Epiretinal membranes were termed "typical tractional ERM" if presenting with contractive properties, or "atypical epiretinal tissue" if presenting as epiretinal material of homogeneous medium reflectivity without contractive properties. Integrity or discontinuity of the inner and outer segment (IS/OS) and the external limiting membrane (ELM) was evaluated by differentiating between "defect present" and "defect absent." RESULTS: In eyes with LMH, atypical epiretinal tissue presented in 29%, typical tractional ERMs were seen in 57%, and a combination of both in 14%. In contrast, eyes with MPH rarely presented atypical epiretinal tissue, and typical tractional ERMs were found in 89%. Comparing cases with LMH, eyes with atypical epiretinal tissue showed significantly more defects of the IS/OS and the ELM than eyes with typical tractional ERM. Both IS/OS and ELM defects correlated with a significant lower best-corrected visual acuity. Defects of the IS/OS were seen in 41% of LMH and 11% of MPH. Defects of the ELM revealed in 27% of LMH and in 11% of MPH. Operated eyes with disrupted IS/OS but intact ELM had significant better best-corrected visual acuity than eyes with defects in both layers. CONCLUSION: Atypical epiretinal tissue is related to the presence of photoreceptor layer defects and to poor visual acuity. It seems that integrity of the ELM is most important for functional recovery after surgery in both LMH and MPH. The presence of atypical epiretinal tissue in eyes with LMH may represent differences in the pathogenesis compared with MPH, and might have therapeutic implications for the proceeding with macular surgery in selected cases.

Hyalocytes in idiopathic epiretinal membranes: a correlative light and electron microscopic study.

PURPOSE: To describe characteristics of epiretinal cells at the vitreoretinal interface by correlative light and electron microscopy (CLEM). METHODS: Epiretinal membrane (ERM) specimens and internal limiting membrane (ILM) specimens were harvested by sequential peeling during vitrectomy from 27 eyes with idiopathic epiretinal gliosis, and processed for CLEM. Intraoperatively, the presence of posterior vitreous detachment (PVD) was documented. We used anti-vimentin, anti-α-smooth muscle actin (α-SMA), and anti-CD45 as primary antibodies. A fluorescein-tagged immunonanogold cluster was used as secondary antibody and visualized under the fluorescence and transmission electron microscope. RESULTS: We demonstrated CD45-positive cells specifically labelled at their plasma membranes with ultrastructural features known for hyalocytes, such as oval nucleus with marginal chromatin, vacuoles, dense granules, and thin cytoplasmic protrusions. CD45-positive cells were mostly located on a thick layer of native vitreous collagen. They were covered by newly formed collagen strands with multilayered proliferation of myofibroblasts. We also demonstrated immunoreactivity for vimentin and alpha-SMA. Cell fragments with positive labelling for α-SMA and vimentin were not only found on the vitreal side of the ILM, but also on the retinal side. CONCLUSIONS: By CLEM, the majority of CD45-positive cells in epiretinal cell proliferation were characterized as hyalocytes. In the context of anomalous PVD and vitreoschisis, ultrastructural features and topographic localization of hyalocytes suggest that these cells play a significant role in ERM formation. CLEM enables a more accurate characterization of epiretinal cell proliferation, and therefore, contributes to a better understanding of the pathogenesis of diseases at the vitreoretinal interface.

Morphological and Clinical Characterization of Foveal Bulge Sign Three Years After Retinal Detachment Repair: A Longitudinal Prospective Evaluation.

Purpose: To evaluate optical-coherence-tomography (OCT)-morphological and clinical parameters three years after primary rhegmatogenous retinal detachment (RRD) repair surgery and the role of postoperative foveal bulge sign. Methods: Of the 32 initially enrolled patients with primary RRD 20 (14 fovea-on, 6 fovea-off) completed this prospective study. Preoperatively and 3 years after surgery best corrected visual acuity (BCVA) and OCT including macula status, central retinal thickness (CRT), central choroidal thickness (CCT), thickness of each single retinal layer, integrity of cone-interdigitation-zone (CIZ) and ellipsoid zone (EZ), and presence of foveal bulge were evaluated. Results: Preoperatively fovea-off RRD patients show significantly thinner CCT, inner nuclear layer (INL) and inner plexiform layer (IPL) compared to fovea-on RRD patients, whereas only IPL and INL were significantly thicker compared to the fellow eye. Three years after surgery this thickening recovered. Final BCVA did not differ statistically significantly between fovea-off and fovea-on patients, no difference in CIZ-integrity or presence of foveal bulge was observed. Presence of foveal bulge at 3 years showed significantly better final BCVA and was associated with intact preoperative CIZ-integrity and postoperative EZ- and CIZ-integrity. The preoperative fovea status showed no correlation to the postoperative presence of foveal bulge. Conclusion: Three years after RRD repair retinal layers show similar thickness. The presence of foveal bulge is associated with better final BCVA. Sufficient pre- and postoperative CIZ-integrity as well as postoperative CIZ-integrity seem to be strongly associated with the restoration of foveal bulge. No correlation was found with the preoperative macular status or BCVA.

We analyzed long-term optical-coherence-tomography changes after rhegmatogenous retinal detachment repair. Three years after rhegmatogenous retinal detachment repair retinal layers show similar thickness and visual acuity did not differ between fovea off and fovea on patients. The restoration of foveal bulge sign was the most important factor for good postoperative visual acuity. Integrity of specific retinal layers, such as the ellipsoid zone and cone-interdigitation-zone, seems to be strongly associated with the restoration of the foveal bulge.