Correlative Microscopy of Lamellar Hole-Associated Epiretinal Proliferation

Abstract

Purpose. To describe morphology of lamellar hole-associated epiretinal proliferation (LHEP) removed from eyes with lamellar macular holes (LMH). Methods. Based on optical coherence tomography data, 10 specimens of LHEP were removed from 10 eyes with LMH during standard vitrectomy. Specimens were prepared for correlative light and electron microscopy (CLEM) using an immunonanogold particle of 1.4 nm diameter that was combined with a fluorescein moiety, both having been attached to a single antibody fragment. As primary antibodies, we used antiglial fibrillary acidic protein (GFAP), anti-CD45, anti-CD64, anti-α-smooth muscle actin (α-SMA), and anticollagen type I and type II. Results. In LHEP, GFAP-positive cells possess ultrastructural characteristics of fibroblasts and hyalocytes. They represent the major cell types and were densely packed in cell agglomerations on vitreous collagen strands. Epiretinal cells of LHEP rarely demonstrated contractive properties as α-SMA-positive myofibroblasts were an infrequent finding. Conclusion. CLEM indicates that epiretinal cells in LHEP might originate from the vitreous and that remodelling processes of vitreous collagen may play an important role in pathogenesis of eyes with LMH.

Lamellar Macular Hole: State of the Art

“…Firstly, CD45, CD64, and GFAP were the most frequent cell markers found in both types of ERM, C ERM and A ERM, suggesting that hyalocytes and glial cells play an important pathogenic role [3, 7, 56-60]. Otherwise, cellular retinaldehyde-binding protein was positive only in a few samples, supporting the hypothesis that RPE cells are not implicated.…”

“…Immunohistochemistry has been widely used to understand the distribution and localization of biomarkers: glial fibrillary acidic protein (GFAP), a marker of the intermediate filaments of glial cells; cellular retinaldehyde-binding protein, a marker of glial cells and RPE cells; neurofilament, a marker of retinal ganglion cells; α-smooth muscle actin (α-SMA), an antibody to intracellular actin filaments; CD45 and CD64, markers of hyalocytes; and CD68, a marker of macrophages/microglia [3, 7]. …”

“…Otherwise, cellular retinaldehyde-binding protein was positive only in a few samples, supporting the hypothesis that RPE cells are not implicated. The studies of CD68 antibodies and neurofilaments produced contrasting results [3, 7, 56-60]. …”

“…Visual acuity may be correlated with ERM type: A ERM is associated with lower visual acuity than C ERM. Figure 3a shows the comparison of BCVA between LMH associated with C ERM and LMH associated with A ERM ( p < 0.0001) [3, 4, 7-11, 22]. The difference in visual acuity may be explained by a greater involvement of outer intraretinal layers ( p = 0.0003) and of the ellipsoid zone and external limiting membrane (ELM), and by a lower residual foveal thickness in LMH associated with A ERM than in LMH associated with C ERM ( p = 0.00001) [3, 4, 8-10, 22] (Fig.…”

“…Moreover, immunohistochemical and microscopic studies have shown differences in cell composition of ERMs, identifying two types of ERM. This fact raised the question as to whether there are two distinct clinical entities or two different stages of the same disorder [3, 7-9, 12, 13]. The different names adopted for LMH and the associated ERMs are the result of disagreement about the final definition of this disorder [2, 3, 5-9, 14].…”