Local Recurrence in Choroidal Melanomas following Robotic-Assisted Radiosurgery (CyberKnife).

Introduction: Tumor recurrence in choroidal melanoma has been associated with decreased overall survival due to metastatic spreading. To detect risk factors of local recurrence and side effects, we analyzed tumor planning and treatment parameters in patients with recurrence of choroidal melanoma after treatment with robotic-assisted radiosurgery (CyberKnife). Methods: Six hundred ninety-four patients treated with CyberKnife between 2005 and 2019 were retrospectively reviewed. Age, gender, best-corrected visual acuity, tumor height, and diameter were recorded. Treatment planning and radiation doses were reviewed. Salvage therapy, overall survival, metastasis, and complications were recorded. Results: Seventy-four patients showed local recurrence. Local recurrence occurred after 42.1 months post CyberKnife treatment (mean; range: 5-100 months). Fourteen out of 74 patients (18.9%) died during follow-up. Recurrence treatment included enucleation in 51 patients (68.9%) and radiosurgery in 19 patients (25.7%). Treatment planning without contrast medium MRI, radiation dose of less than 21 Gy, and insufficient margin delineation were identified as risk factors incrementing local control. Discussion: Robotic-assisted radiosurgery (CyberKnife) is a suitable treatment option for large choroidal melanoma up to 12 mm. Patients with significantly better visual acuity received repeat CyberKnife treatment as salvage therapy and showed an eye retention rate of 81%.

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.

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.

Comparison of Vitreomacular Interface Changes in Myopic Foveoschisis and Idiopathic Epiretinal Membrane Foveoschisis.

PURPOSE: To compare characteristics of the vitreomacular interface (VMI) in myopic foveoschisis (mFS) and idiopathic epiretinal membrane foveoschisis (iERM-FS), and to correlate with optical coherence tomography and clinical data. DESIGN: Clinicopathologic study. METHODS: Epiretinal membrane and internal limiting membrane (ILM) specimens were removed from eyes with mFS (5 eyes) and iERM-FS (5 eyes). Harvested tissue was processed for immunocytochemistry and prepared by ultrathin series sectioning for transmission electron microscopy. Cell and collagen compositions were compared and correlated with clinical data. RESULTS: All eyes presented fibrocellular membranes irrespective if associated with mFS or iERM-FS. Cell and collagen types and distribution on the vitreal side of the ILM were similar in both groups, consistent with presence of tractional membranes on optical coherence tomography images. Immunostaining of all specimens were positive for glial cells, microglia, and hyalocytes. Electron microscopy revealed evidence of epiretinal cell multilayers with masses of vitreous collagen and signs of vitreous remodeling in both groups. Three eyes with mFS but none of the eyes with iERM-FS showed massive thinning of the ILM with prominent retinal undulations and presence of retinal nerve fiber layer fragments. CONCLUSION: Whereas fibrocellular components of premacular tissue in mFS are similar to iERM-FS, pathologic abnormalities of the ILM were exclusively present in high myopia. Although peeling of the ILM appears important to completely remove tractional components of the VMI, histopathologic findings emphasize the risk for retinal damage in these highly myopic eyes, indicating that individual preoperative assessment and modification of surgical techniques require further investigation.

Premacular Cells as Source of Neurotrophic Factors in Idiopathic Macular Holes.

Purpose: To describe the presence of neurotrophic growth factors and histopathologic characteristics of internal limiting membrane (ILM) specimens obtained from large idiopathic full-thickness macular holes (FTMH). Methods: In 24 eyes of 24 patients with FTMH of diameter >400 µm, ILM specimens were harvested directly at the edge surrounding the macular hole during vitrectomy with peeling. We performed interference and phase contrast microscopy of flat mounts followed by immunostaining and transmission electron microscopy. Primary antigens directed against neurotrophic growth factors as well as antigens to glial and ganglion cells were used. Topographic relationship of cells and collagen was demonstrated by serial ultrathin sectioning. Results: Immunofluorescence microscopy demonstrated the presence of glial-derived neurotrophic factor and ciliary neurotrophic factor. Expression of vimentin, glial fibrillary acidic protein (GFAP), neurofilament, calretinin, and melanopsin was seen positive too. Cellular retinaldehyde-binding protein was seen positive in half of the specimens. Co-localisation of anti-GFAP as well as anti-vimentin with neurotrophic factors was found. Electron microscopy revealed cells exclusively on the vitreal side of the ILM. Cell fragments on the retinal side were rarely seen. Conclusion: In large FTMH, ILM specimens present positive immunolabelling of neurotrophic factors. The co-localization with macroglial cell markers suggests a premacular cell composition as a source of the neurotrophic factors. Ultrastructurally, premacular cells were found on the vitreal side of the ILM and not within the collagen network of the ILM itself.

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.

Comparison of Surgically Excised Premacular Membranes in Eyes with Macular Pucker and Proliferative Vitreoretinopathy.

PURPOSE: To describe and compare characteristics of premacular membranes in idiopathic macular pucker (iMP) and proliferative vitreoretinopathy (PVR) using immunofluorescence and transmission electron microscopy. MATERIALS AND METHODS: For immunocytochemical and ultrastructural analyses, premacular membranes were harvested during vitrectomy from 16 eyes with iMP and 12 eyes with PVR. All specimens were processed as flat mounts for phase-contrast and fluorescence microscopy. We used 19 different primary antibodies such as anti-α-smooth muscle actin (α-SMA), anti-integrin-αv, anti-galectin, anti-IBA-1, anti-EMMPRIN (CD147), anti-ricinus (RCS) and anti-collagen-type I. Eight of 28 eyes were also prepared for transmission electron microscopy. RESULTS: In all eyes with iMP and PVR, positive immunoreactivity of integrins, especially αvß3 was found. There was also a strong staining of anti-α-SMA, anti-galectin, anti-EMMPRIN, anti-RCS, anti-IBA1 and anti-collagen-type I. Transmission electron microscopy showed that premacular membrane of iMP composed of myofibroblasts, glial cells and fibroblasts. In eyes with PVR, retinal pigment epithelial cells and myofibroblasts were seen as predominant cell types. CONCLUSION: Premacular membranes of iMP and PVR presented with similarities in cell distribution and immunoreactivity, but showed differences in cell composition. Herein, we demonstrate immunocytochemical characteristics involved in fibrotic processes. Cell transdifferentiation into myofibroblasts represents an important process in pathogenesis of both entities. In order to address future anti-fibrotic treatment strategies, we emphasize that both fibrotic diseases share distinct immunocytochemical and ultrastructural features.

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.

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.