Sir,

There are only rare reports showing the presence of α-smooth muscle actin (α-SMA)staining-positive cells in internal limiting membrane (ILM) specimens removed during idiopathic macular hole surgery, and in these studies, specimens have been evaluated by means of light or scanning electron microscopy.1, 2, 3 To the best of the authors’ knowledge, identification and localisation of α-SMA microfilaments by confocal microscopy in the ILM surrounding the borders of a macular hole have not been previously described.

Case report

A 68-year-old woman underwent vitrectomy for stage 4 idiopathic macular hole. Preoperative evaluation of the macular hole biomicroscopically and imaging with optical coherence tomography showed the absence of an epiretinal membrane (Figure 1b). Triamcinolone–acetonide was used for visualising the posterior cortical vitreous. After vitrectomy, the ILM was stained with Brilliant blue (BB) dye, then peeled off using intraocular forceps. The part of the ILM around the edge of the macular hole was not stained (Figure 1a). Two separate ILM specimens were obtained during surgery; the first (specimen A) around the macular hole and the second (specimen B) 1-disc diameter away from the macular hole up to the temporal vascular arcades and the optic disc.

Figure 1
figure 1

Imaging of the macular area. (a) Intraoperative visualisation of the macular hole and the internal limiting membrane (ILM) after staining with Brilliant blue dye. Note that the area around the edge of the macular hole is not stained blue, suggesting the presence of cells covering the ILM even in the absence of a formed epiretinal membrane (ERM). (b) Macular optical coherence tomography shows a full-thickness macular hole and confirms the absence of any ERM.

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The ILM specimens were studied immunohistochemically using confocal microscopy after labelling with antibodies to α-SMA. ILM specimen A was stained positive for α-SMA (Figure 2a–c), whereas specimen B failed to stain positive for α-SMA (Figure 2d).

Figure 2
figure 2

Confocal microscopy imaging of the internal limiting membrane (ILM) in a stage 4 idiopathic macular hole. ILM peeled around the macular hole (specimen A). (a) Fibronectin is an extracellular glycoprotein that is distributed throughout the ILM of the human retina. After immunostaining with antibodies to fibronectin, the ILM acquired a homogenous nonspecific staining (green) that was used as a marker of ILM surface. (b) α-smooth muscle actin (α-SMA), a contractile intracellular protein arranged in microfilaments, is a marker of myofibroblasts. α-SMA is coloured red. Colocalisation image showing simultaneously the ILM (in green) and bundles of actin microfilaments (in red) after immunostaining with antibodies to both fibronectin and α-SMA. Note the dense network of interweaving α-SMA microfilaments. (c) Part of the same ILM specimen showing characteristic red-coloured actin microfilaments (white arrows) on its surface (coloured green). Note also the presence of some grains of triamcinolone–acetonide (arrowhead) deposited directly on the surface of the ILM. (d) Confocal microscopy imaging of the ILM (specimen B) peeled 1–2 disc diameters away from the macular hole. Both antibodies to fibronectin and α-smooth muscle actin (α-SMA) have been used. Note that this ILM specimen does not contain any α-SMA filaments as it only stains in green.

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Comment

Vitreofoveal traction has been proposed as the main mechanism for early stage (1 and 2) idiopathic macular hole formation.4 Cellular migration and proliferation with secondary contraction on the ILM might lead to a further progression of the macular hole even after posterior vitreous detachment has occurred (stages 3 and 4) and keep the macular hole open, with stage 4 macular holes having the largest area of cellular migration around the macular hole.5 Our case clearly shows that cells on the ILM around stage 4 macular hole contain bundles of actin microfilaments and therefore have contractile properties. The presence of cells should be suspected when the ILM staining around the macular hole fails to stain with BB, even when OCT does not show any ERM, and should guide the surgeon in favour of ILM removal. These cells may arise from retinal Müller cells that have the capacity to change their phenotype to transdifferentiate into myofibroblast-like cells and express α-SMA.6

Our findings suggest that ILM may serve as a scaffold for cellular migration and proliferation and contribute to the pathogenesis of stage 4 macular hole formation. Surgical peeling of the ILM in stage 4 macular holes even without evidence of an epimacular membrane would allow the removal of contractile myofibroblasts with consequent elimination of any tangential tractional forces around the macular hole, thus leading to its successful closure. This is in accordance with a large study showing that ILM peeling is of particular benefit for obtaining anatomical hole closure in stage 3 and 4 macular holes.7