Natural History and Surgical Outcomes of Lamellar Macular Holes.

PURPOSE: To assess the natural history and surgical outcomes of lamellar macular holes (LMHs). DESIGN: Retrospective and consecutive case series. SUBJECTS: Patients with LMHs from multiple tertiary care centers. METHODS: Clinical charts and OCT scans were reviewed. MAIN OUTCOME MEASURES: The visual acuity (VA) changes and the occurrence rate of full-thickness macular hole (FTMH) were studied in both groups. Within the operated group, factors associated with 6-month VA and development of FTMH were explored. RESULTS: One hundred seventy-eight eyes were included, of which 89 were monitored and 89 underwent surgery. In the observation group, the mean VA decreased from 0.25 ± 0.18 to 0.28 ± 0.18 logarithm of the minimum angle of resolution (logMAR; P = 0.13), with 14 eyes (15.7%) that lost ≥ 0.2 logMAR VA, after 45.7 ± 33.3 months. Nine eyes (10.1%) spontaneously developed an FTMH. In the operated group, the mean VA increased from 0.47 ± 0.23 to 0.35 ± 0.25 logMAR at 6 months (P < 0.001) and 0.36 ± 0.28 logMAR (P = 0.001) after 24.1 ± 30.1 months. By multivariate analysis, better baseline VA (P < 0.001), the presence of an epiretinal membrane (P = 0.03), and the peeling of the internal limiting membrane (ILM; P = 0.02), with a greater effect of ILM perihole sparing, were associated with a greater 6-month VA. Perihole epiretinal proliferation sparing was associated with a better postoperative VA by univariate analysis (P = 0.03), but this was not significant by multivariate analysis. Eight eyes (9.0%) developed a postoperative FTMH. Using Cox proportional hazard ratios [HRs], pseudophakia at baseline (HR, 0.06; 95% confidence interval [CI], 0.00-0.75; P = 0.03) and peeling of the ILM (HR, 0.05; 95% CI, 0.01-0.39; P = 0.004) were protective factors, while ellipsoid zone disruption (HR, 10.5; 95% CI, 1.04-105; P = 0.05) was associated with an increased risk of FTMH. CONCLUSION: Observed eyes with LMH experienced, on average, progressive VA loss. Patients with LMH and altered vision may benefit from surgery. Internal limiting membrane peeling, with perihole ILM sparing, represents a crucial step of the surgery associated with a greater VA and a lower risk of postoperative FTMH. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Optimal Patient Adherence and Long-Term Treatment Outcomes of Neovascular Age-Related Macular Degeneration in Real-Life.

PURPOSE: To report on long-term real-life outcomes of anti-vascular endothelial growth factor (anti-VEGF) therapy in neovascular age-related macular degeneration (nAMD) with optimal patient adherence. METHODS: For this retrospective monocenter study, we identified 3217 eyes of 2793 patients that received a minimum of three intravitreal anti-VEGF injections for nAMD therapy between 2006 and 2014 at the University Eye Hospital Munich. From those, we included eyes with treatment-naïve nAMD, follow-up (FU) of ≥60 months and continuous adherence during FU. Primary measures were corrected visual acuity (VA), number of injections and visits as well as treatment regimen. RESULTS: We included 161 eyes of 125 patients with a mean FU of 8.0 ± 2.3 years. Mean VA at baseline was 60.1 letters (Snellen equivalent, 20/63). After the third year, mean VA declined constantly by 2-3 letters per year. After 5 and 8 years, 26.1% and 42.1% had lost at least 3 lines from baseline. Mean cumulative number of injections was 5.3 after the first year, and 23.9, 38.1, 48.5 after 5, 8, and 10 years. "Treat and extent" regimen with higher injection frequency correlated with better function. At time of last FU, 69.8% of eyes were under active treatment. Eyes with ≥70 letters at baseline correlated with better VA at the end of FU. CONCLUSIONS: Despite optimal patient adherence, visual function declined progressively in real-life nAMD therapy over long-term. The highest impact on treatment success is given by an early treatment start with individual but intensive anti-VEGF therapy.

CENTRAL POSTERIOR LENS CAPSULE USED INSTEAD OF AN INVERTED INTERNAL LIMITING MEMBRANE FLAP: A CLINIC-PATHOLOGICAL CASE REPORT.

PURPOSE: To present immunocytochemical characterization of a surgically excised central posterior lens capsule (PLC) that was transplanted to close a secondary refractory full-thickness macular hole (FTMH) as an epiretinal flap. For comparison, tissue of both an unaffected internal limiting membrane and unexposed PLC was processed. METHODS: Clinical-pathological case report. RESULTS: We report of a 38-year-old patient who underwent pars plana vitrectomy (PPV) with PLC tissue for patching secondary FTMH and silicone oil tamponade after tractional retinal detachment. The PLC was peeled off during a vitrectomy 1 year after positioning. For immunocytochemistry, the removed PLC was prepared as flat mount and showed a positive immunofluorescence of the Müller cells marker glutamine synthetase and for vimentin. The microglia marker IBA and the neuroprotective neurotrophic marker glia cell-derived neurotrophic factor were tested positive too. There was no immunoreactivity of cellular retinaldehyde-binding protein and glial fibrillary acidic protein. In comparison, tissue of a control internal limiting membrane that was obtained during standard FTMH surgery showed few single cells that were likewise positive for glutamine synthetase, glia cell-derived neurotrophic factor, and IBA. The control specimen of unexposed PLC showed rarely cells that were without positive immunostaining for the tested markers. CONCLUSION: Our analysis revealed positive immunoreactivity of macroglia and microglia cells of the PLC tissue that was used to patch a refractory FTMH. Similar immunostaining of PLC material and internal limiting membrane suggests the PLC transplantation as an alternative treatment option for refractory FTMH.

Impact of Sub-Retinal Fluid on the Long-Term Incidence of Macular Atrophy in Neovascular Age-related Macular Degeneration under Treat & Extend Anti-Vascular Endothelial Growth Factor Inhibitors.

Sub-retinal fluid (SRF) has been discussed as a protective factor against macular atrophy in eyes with neovascular age-related macular degeneration (nAMD).To gauge the impact of SRF on macular atrophy, a database of 310 nAMD eyes was screened for eyes manifesting an SRF-only phenotype under treat & extend anti-VEGF treatment, defined as nAMD expressing CNV exudation beyond the three monthly anti-VEGF loading doses by SRF only without any signs of exudative intra-retinal fluid (IRF) for ≥3 years. Incidence of macular atrophy and treatment responses were evaluated on multimodal imaging, including optical coherence tomography (OCT), blue autofluorescence (BAF) and near-infrared (NIR) confocal scanning laser ophthalmoscopy and fluorescence and indocyanine green angiography (FAG/ICGA). In total, 27 eyes (8.7%) of 26 patients with a mean follow-up of 4.2 ± 0.9 (3-5) years met the inclusion criteria. Mean age was 72 ± 6 (range: 61-86) years. The SRF only phenotype was seen from baseline in 14 eyes (52%), and in 13 eyes (48%) after a mean 1.0 ± 1.3 (1-3) injections. In years 1 to 5, mean 7.5, 5.9, 6.1, 6.1 and 7.0 anti-VEGF injections were given (p = 0.33). Cumulative macular atrophy incidence was 11.5% at year 1, 15.4% throughout years 2 to 4, and 22.4% at year 5. In conclusion, eyes manifesting activity by SRF only in treat & extend anti-VEGF regimen for nAMD seem to exhibit rather low rates of macular atrophy during long-term follow-up. SRF might be an indicator of a more benign form of nAMD.

Lamellar hole-associated epiretinal proliferation presenting with perifoveal exudative vascular anomalous complex.

PURPOSE: Spectral-domain optical coherence tomography (SD-OCT) commonly reveals lamellar-hole-associated epiretinal proliferation (LHEP) as an avascular homogenous layer of premacular material with medium reflectivity, as recently described in various traction maculopathies, mostly in lamellar macular holes (LMH). We have used multimodal imaging to examine a patient suffering from unilateral advanced atrophic LMH presenting LHEP with perifoveal exudative vascular anomalous complex (PEVAC) and intra-LHEP edema fluctuating under anti-vascular endothelial growth factor (anti-VEGF) therapy. OBSERVATION: A 77-year-old male presented with decreased vision in the left eye attributable to longstanding LMH. He complained of worsening symptoms for six months. Whereas SD-OCT showed classic tractional epiretinal gliosis in the right eye, the left eye exhibited atrophic LMH and a significant amount of LHEP containing hyperreflective round lesions and hyporeflective cystoid spaces. Fluorescein/indocyanine green angiography demonstrated PEVAC with large anomalous vessels and exudation. OCT angiography revealed abnormal vessels originating from the deep retinal plexus. After anti-vascular endothelial growth factor (anti-VEGF) therapy, the intraretinal edema seemed to decrease. CONCLUSIONS AND IMPORTANCE: Perifoveal exudative vascular anomalous complex can occur in eyes with advanced LMHs causing edema inside LHEP. Pathologic vessels appear to originate from the deep retinal plexus. Given that LHEP formation is proposed to be a glial-cell-driven process, Müller cells may play a decisive role in the pathogenesis of the presented vascular malformation. Because of spontaneous fluctuation of the associated edema, the role of anti-VEGF remains questionable, while a functional response to therapy might be limited according to the progressive atrophic lamellar defect with intraretinal tissue loss.

DEVELOPMENT AND PROGRESSION OF A LAMELLAR MACULAR HOLE WITH LAMELLAR HOLE-ASSOCIATED EPIRETINAL PROLIFERATION.

PURPOSE: To describe evolution and progression of a lamellar macular hole (LMH) with "lamellar hole-associated epiretinal proliferation (LHEP)" using spectral-domain optical coherence tomography and fundus autofluorescence. METHODS: Observational case report. RESULTS: We report on a 63-year-old male patient demonstrating a complete history of LMH development with LHEP occurring during a follow-up period of 8 years. Presenting with a normal foveal contour and attached posterior vitreous at first visit, an LMH developed shortly after incomplete posterior vitreous detachment with vitreopapillary adhesion. On spectral-domain optical coherence tomography images, progression of the LMH including enlargement of the intraretinal cavitation and decrease in the retinal thickness were documented. An increase of LHEP was first documented 6 months after LMH evolution. One month after cataract surgery and 6 years after the first visit, a full-thickness macular hole developed that closed spontaneously after 4 weeks. Localization of LHEP had moved into the foveal defect toward the outer retinal layers. Thereafter, the LMH was stable, and the patient presented with a visual acuity of 20/25. CONCLUSION: Proper follow-up time is important for studying eyes with an LMH. Epimacular cell proliferation shows progression over time that appears to be associated with morphologic changes of the LMH including shape of the lamellar defect, amount of LHEP, and contractive properties of epiretinal tissue. The presence of LHEP was documented shortly after posterior vitreous detachment.

Vitrectomy for Diabetic Macular Edema: Optical Coherence Tomography Criteria and Pathology of the Vitreomacular Interface.

PURPOSE: To correlate spectral-domain optical coherence tomography (SDOCT) criteria and clinical data with pathology of the vitreomacular interface (VMI) in eyes with diabetic macular edema (DME). DESIGN: Retrospective cross-sectional study and laboratory investigation. METHODS: We included specimens of 27 eyes of 26 patients with center-involved DME that underwent vitrectomy with peeling of the internal limiting membrane (ILM). Selection of specimens was consecutive and in retrospect using our register of the Vitreoretinal Pathology Unit. Clinical data and SDOCT examinations were correlated to immunocytochemistry and transmission electron microscopy. Classification of DME comprised sponge-like diffuse retinal thickening, cystoid macular edema, and serous retinal detachment. VMI was evaluated for presence of epiretinal membrane (ERM) and thickened vitreous cortex (tVC). RESULTS: ERMs and tVC were found in all DME types. Diffuse DME showed tVC more often than cystoid DME. Hyalocytes, contractile myofibroblasts, glial cells, matrix metalloproteinases-2 and -9, and collagen type I, II, and III were positive tested irrespective of DME type. There were no significant cell fragments at the retinal side of the ILM. Visual acuity improved in the majority of cases and macular thickness decreased significantly during mean follow-up of 17 ± 10 months. CONCLUSIONS: All eyes presented pathologic VMI changes irrespective of the OCT classification of DME type or presence of ERM. Composition of fibrocellular membranes at the VMI indicated remodeling of vitreous cortex and transdifferentiation of hyalocytes into myofibroblasts. Our findings might argue for an early surgical intervention in eyes with DME irrespective of the presence of traction formation imaged by SDOCT.

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.

Wound Healing, Inflammation, and Corneal Ultrastructure After SMILE and Femtosecond Laser-Assisted LASIK: A Human Ex Vivo Study.

PURPOSE: To assess the wound healing, inflammation, and tissue ultrastructure in the human corneal stroma after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). METHODS: Sixteen corneoscleral discs of 16 human donors unsuitable for corneal transplantation were obtained from an eye bank. Eight eyes underwent SMILE with -5.00 diopters (D) of myopic correction; in 3 of them the lenticule was not extracted. Further 5 donor corneas were subjected to FS-LASIK with -5.00 D ablation, and 3 eyes served as the control group without surgical intervention. Postoperatively, specimens were incubated in organ culture medium for 72 hours before being subjected to immunofluorescence staining for CD11b, Ki67, fibronectin, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay, and high-magnification scanning electron microscopy. RESULTS: Keratocyte apoptosis, keratocyte proliferation, and infiltration of immune cells were generally mild and comparable between FS-LASIK and SMILE (irrespective of surgical lenticule extraction). By staining for fibronectin, we observed a trend toward milder fibrotic response in the corneal stroma after SMILE than after FS-LASIK. On the contrary, scanning electron microscopy analysis revealed a smoother, more regular ultrastructural appearance of the residual corneal bed after FS-LASIK. CONCLUSIONS: Corneal stromal wound healing after SMILE and FS-LASIK was virtually identical with respect to keratocyte proliferation and apoptosis in the human donor eye model. Although reactive fibrosis adjacent to the laser application site appeared less marked after SMILE, the stromal bed after LASIK exhibited a smoother surface texture. [J Refract Surg. 2018;34(6):393-399.].

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.

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.

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.

Increase in lens capsule stiffness caused by vital dyes.

PURPOSE: To assess potential changes in lens capsule mechanical properties after staining with brilliant blue, indocyanine green (ICG), and trypan blue. SETTING: Department of Ophthalmology and Applied Physics and Center for NanoScience, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Fifteen unstained lens capsules were dissected into 7 wedge-shaped parts. Three fragments were stained with brilliant blue 0.025%, ICG 0.05%, and trypan blue 0.06%, respectively, for 1 minute. Another 3 specimens were additionally illuminated using a standard light source. The seventh part served as an untreated control. All specimens were analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 µm × 10 µm were chosen, and stiffness was determined using AFM in a force spectroscopy mode. The force curves were performed with a data rate of 5000 Hz. RESULTS: Staining of the samples resulted in an increase in tissue stiffness (brilliant blue: P<.001; ICG: P<.01; trypan blue: P<.05). Additional illumination after staining further increased tissue stiffness, but not significantly. Mean increase in the relative elasticity values were 1.61 ± 0.15 (SD) for brilliant blue, 2.04 ± 0.21 for brilliant blue with illumination, 1.63 ± 0.22 for ICG, 2.01 ± 0.22 for ICG with illumination, 1.23 ± 0.11 for trypan blue, and 1.39 ± 0.11 for trypan blue with illumination. In relation to unstained tissue, the relative elasticity of the stained tissue increased 1.2-fold after illumination. CONCLUSION: Staining significantly increased the mechanical properties of the human lens capsule. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Quantification of contrast recognizability in sequential epiretinal membrane removal and internal limiting membrane peeling in trypan blue-assisted macular surgery.

PURPOSE: To evaluate the selectivity and strength of intraoperative trypan blue staining during removal of epiretinal membranes (ERMs) and the internal limiting membrane. METHODS: Based on intraoperative videos, 51 consecutive chromovitrectomies in 51 patients with macular holes, macular pucker, vitreomacular traction syndromes, or persistent macular edema were retrospectively studied. Fifteen subjects underwent trypan blue, 14 indocyanine green, and 22 brilliant blue G chromovitrectomy. The main outcome measure was the color contrast between stained internal limiting membrane or ERM and the underlying unstained tissue by means of objective, quantitative, semiautomated chromaticity difference measurements. RESULTS: Trypan blue stains both ERM and the internal limiting membrane (average chromaticity scores 8.51 and 7.09, respectively; P = 0.48). Internal limiting membrane chromaticity scores were similar for trypan blue (7.09) and brilliant blue G (6.81; P = 0.71) but clearly higher for indocyanine green (15.81; P = 2.45 × 10). CONCLUSION: Under the premises of our study, trypan blue stains both ERM and the internal limiting membrane. Trypan blue's staining capacity of the internal limiting membrane is similar to that of brilliant blue G but significantly inferior compared with indocyanine green. Trypan blue, thus, represents a useful vital dye for chromovitrectomy, particularly in the presence of ERM, where it allows a sequential approach.

Epiretinal cell proliferation in macular pucker and vitreomacular traction syndrome: analysis of flat-mounted internal limiting membrane specimens.

PURPOSE: To describe new details of epiretinal cell proliferation in flat-mounted internal limiting membrane specimens. METHODS: One hundred nineteen internal limiting membrane specimens were removed en bloc with epiretinal membranes from 79 eyes with macular pucker (MP) and 40 eyes with vitreomacular traction syndrome. Intraoperatively, posterior vitreous detachment was assessed as complete or incomplete. Whole specimens were flat-mounted on glass slides and processed for interference and phase-contrast microscopy, cell viability assay, and immunocytochemistry. RESULTS: Mean cell viability percentage was higher in MP than in vitreomacular traction syndrome. Two cell distribution patterns were found. Anti-CD163 labeling presented predominantly in MP with complete posterior vitreous detachment. CD45 expression was similar in all groups of diagnosis. Anti-glial fibrillary acidic protein (GFAP) labeling was found in MP irrespective of the extent of posterior vitreous detachment. Alpha-SMA (α-smooth muscle actin) labeling was mainly presented in MP with incomplete posterior vitreous detachment and in vitreomacular traction syndrome. Simultaneous antibody labeling included GFAP/CD45, GFAP/CD163, CD163/CD45, and CD163/α-SMA. CONCLUSION: Hyalocytes constitute a major cell type of epiretinal cell proliferation in eyes with MP and vitreomacular traction syndrome. Glial cells, notably retinal Muller cells, are involved as well. It appears that transdifferentiation of cells in vitreomacular traction might be more frequent than previously thought and that those cells possess a greater variability of immunocytochemical properties than expected.

Lamellar macular hole: a clinicopathologic correlation of surgically excised epiretinal membranes.

PURPOSE: To correlate clinical and optical coherence tomographic (OCT) features with morphologic and immunohistochemical findings of epiretinal membranes (ERMs) in lamellar macular holes (LMHs). METHODS: Nineteen specimens were removed from 19 eyes during vitrectomy for lamellar macular hole with ERM and internal limiting membrane peeling, and were processed for transmission electron microscopy and immunohistochemistry by cross-sectional and flat-mount preparation techniques. By using OCT criteria and intraoperative observations, ERM specimens were divided into two groups: 13 "dense" and 6 "tractional" membranes. Patients' records were reviewed. RESULTS: "Dense" ERMs were seen with abundant clusters of fibrous long-spacing collagen embedded in compactly folded native vitreous collagen strands. Posterior hyaloids were attached to the retina in the majority of cases. Both groups of ERMs showed positive immunoreactivity for glial fibrillic acidic protein and hyalocyte markers. Anti-α-smooth muscle actin labeling was most positive in "tractional" ERMs. Surgery resulted in significant improvement (74%) of visual acuity, with a mean gain of 2 Snellen lines in both groups. All other patients (26%) preserved vision. Three patients (16%) developed a full-thickness macular defect requiring additional surgery. CONCLUSIONS: Morphologic components differ in epiretinal cell proliferations of LMHs. In association with degradation of vitreous collagen, glial cells and hyalocytes seem to play an important role in LMH development. Since functional benefit after surgery was limited and progression to a full-thickness macular defect was a notable complication, we recommend caution in proceeding with surgical intervention. Further investigations are needed to elucidate whether "dense" and "tractional" LMHs represent different disease entities or different stages of one disorder.

Sequential epiretinal membrane removal with internal limiting membrane peeling in brilliant blue G-assisted macular surgery.

PURPOSE: To assess the selectivity of brilliant blue G (BBG) staining by analysing the morphological components of unstained and stained tissue obtained during epiretinal membrane (ERM) removal with internal limiting membrane (ILM) peeling in BBG-assisted macular surgery. METHODS: Twenty-six surgical specimens were removed from 13 eyes with epiretinal gliosis during vitrectomy using BBG for ERM and ILM peeling. We included eyes with idiopathic macular pucker, idiopathic macular hole and vitreomacular traction syndrome. The dye was injected into the fluid-filled globe. Unstained and stained epiretinal tissue was harvested consecutively and placed into separate containers. All specimens were processed for conventional transmission electron microscopy. RESULTS: The first surgical specimen of all eyes showed no intraoperative staining with BBG and corresponded to masses of cells and collagen. The second surgical specimen demonstrated good staining characteristics and corresponded to the ILM in all patients included. In seven eyes, the ILM specimens were seen with minor cell proliferations such as single cells or a monolayer of cells. Myofibroblasts, fibroblasts and astrocytes were present. In five cases, native vitreous collagen fibrils were found at the ILM. In six of the eyes, ILM specimens were blank. CONCLUSION: Our clinicopathological correlation underlines the selective staining properties of BBG. The residual ILM is selectively stained by BBG even when a small amount of cells and collagen adheres to its vitreal side. To reduce the retinal exposure to the dye, the surgeon might choose to remove the ERM without using the dye, followed by a BBG injection to identify residual ILM.

Vital dyes for macular surgery: a comparative electron microscopy study of the internal limiting membrane.

PURPOSE: To analyze and compare the ultrastructure of the retinal cleavage plane of the internal limiting membrane (ILM) removed during dye-assisted macular surgery. METHODS: Ninety-six surgical specimens of the ILM obtained during trypan blue (TB)-, brilliant blue G (BBG)-, bromphenol blue (BPB)-, Chicago blue (CB)-, and indocyanine green-assisted pars plana vitrectomy for idiopathic macular hole and macular pucker were evaluated using transmission electron microscopy. Specimens removed without dye assistance served as controls. RESULTS: Specimens removed after TB-, BBG-, BPB-, and CB-staining presented with significantly less cellular fragments (in size and quantity) at the retinal side of the ILM than specimens after indocyanine green staining. Large cellular fragments and Müller cell endfeet, which were typically present after indocyanine green staining, were not found after TB, BBG, BPB, and CB staining. Comparing all groups, TB was found with less retinal debris than BBG, BPB, and CB. Specimens removed without intravitreal dye administration showed the least retinal fragments that were solitarily distributed at the ILM. CONCLUSION: Trypan blue, BBG, BPB, and CB cause significantly less morphologic changes at the retinal cleavage plane than indocyanine green. Further studies are required to elucidate if presence and amount of retinal cell fragments at ILM specimens correlate with functional deficits.

Idiopathic macular holes: ultrastructural aspects of surgical failure.

PURPOSE: To investigate the ultrastructure of the internal limiting membrane (ILM) and epiretinal tissue in eyes with idiopathic macular holes that were not successfully closed by one operation. METHODS: A second vitrectomy with en bloc removal of the ILM and epimacular tissue was performed in 16 eyes with full-thickness macular holes after surgical failure. The specimens were processed for transmission electron microscopy. In 5 of 16 eyes, specimens of first macular hole surgery were also analyzed. RESULTS: Fibrocellular proliferation at the vitreal side of the ILM was found in all specimens from second vitrectomy. Myofibroblasts and fibroblasts were predominant. Cells were frequently observed as irregular accumulations rather than regular multilayers at the ILM. Masses of newly formed collagen were found distributed between cells and ILM. All specimens from first macular hole surgery were characterized by regular cellular layers and the presence of native vitreous collagen. CONCLUSIONS: Eyes with idiopathic macular holes that were found not to be closed early after the first vitrectomy show massive proliferation of cells and newly formed collagen irregularly distributed at the remaining ILM. After surgical intervention, ILM remnants and collagen may represent a stimulus for the early formation of tangential traction preventing successful macular hole closure.