A full thickness macular hole is a round break involving all layers of the retina, from the internal limiting membrane (ILM) to the outer segments of the photoreceptor layer. The causative factors implicated in macular hole formation are trauma, pathological myopia, laser photocoagulation, choroidal neovascularisation, post-cataract surgery and diabetic retinopathy.12 The pathogenic mechanism for idiopathic macular hole proposed by Johnson et al centers on tangential vitreous traction.1 In traumatic macular hole, the presumed mechanism is concussive effects and residual vitreous traction following incomplete acute vitreous detachment.13 In myopic macular hole, cystic macular degeneration in addition to the anteroposterior vitreous tractions leads to abrupt dehiscence of macular tissue.1
Wendel et al first demonstrated that vitrectomy, posterior cortical vitreous removal and gas tamponade allowed closure of established full-thickness macular hole with improved visual acuity.4 The rationale of this surgery originates in traction being identified as the cause of hole formation and foveal dehiscence, which is responsible for visual deterioration.56 Meta-analysis of the literature suggests that surgery be offered for full- thickness macular hole as early as possible.7 To improve the visual outcome, various adjuvants are used, though the precise role of these adjuvants has not yet been well-established.89101112 The ILM removal has been suggested as a useful adjunct in macular hole surgery.1314 This layer can act as a scaffold for cell proliferation under pathological situations and is responsible for the pathogenesis of disorders that affects the vitreo-retinal interface including epiretinal membrane (ERM), vitreo-macular traction and macular hole.151617
This is a prospective study to describe the ultrastructural features of surgically removed specimens (ILM peel) from different groups of patients and the visual outcome following indocyanine green (ICG)-assisted removal of the ILM in patients with macular hole.
Patients and Methods
This prospective study includes eyes with full thickness macular hole with best-corrected visual acuity (BCVA) equal or worse than 6/12 (0.5) on the Snellen's chart. Detailed preoperative examination including relevant history, BCVA, slit lamp examination, fundus biomicroscopy, indirect ophthalmoscopy and optical coherence tomography (OCT) was done. The parameters noted were age, sex, stage and size (on OCT) of hole, duration of visual symptoms, lens status, presence of posterior vitreous detachment and other factors. Preoperative informed consent was taken from all the patients involved in the study. Surgical success has been defined as flattening of the hole edge and disappearance of sub-retinal fluid (SRF) indicating a closed hole or presence of SRF around the hole indicating open hole.
For comparison of the electron microscopic features of the ILM, four groups were designed and surgery was performed. The four groups were idiopathic macular hole, myopic macular hole, traumatic macular hole and negative controls. Patients who had retinal detachment with proliferative vitreoretinopathy and tractional retinal detachment were included as negative controls. In these patients, removal of the ILM in areas of traction is supposed to decrease the recurrence of proliferative vitreoretinopathy.18
The surgical technique utilised was the same as previously reported.13 The ILM was grasped with pic-forceps (Grieshaber, Alcon Laboratories, Inc., USA). The ILM was peeled so as to cover as much of the area of the macula as possible. Repeat surgery for persistent SRF at the hole edges was done after three months of the first surgery in two cases, one in idiopathic macular hole group and one in myopic macular hole group. In each of these cases, the ILM removed was of a large size. The vitreous cavity was filled with 18% non-expansile volume of sulphur hexafluoride and the sclerotomies were closed with 6-0 Vicryl suture. The patients were advised to follow strict face down positioning for one week. There was a follow-up of patients for a minimum of six months (once in every two weeks for the first three months and then once in every four weeks) and details of BCVA, hole status, lens status, intraocular pressure, presence of iatrogenic break were recorded.
Immediately after peeling, the surgical specimens were fixed in Karnovsky's fixative in 0.1 M phosphate buffer (pH 7.4) for two hours at 4°C. After wash, these were post-fixed in 1% OsO4 for two hours at 4°C, dehydrated in ascending grades of ethyl alcohol and embedded in araldite CY 212. Thin sections (70 nm) were cut with a glass knife and mounted onto nickel grids. They were contrasted with uranyl acetate and lead citrate and viewed under a Philips CM10 transmission electron microscope.
The preoperative and postoperative data of the patients are summarised in Tables 1 and 2. The traumatic cases with cataract had sectoral involvement with clear visual axis. One case (serial # 17 in Table 1) had significant progression in cataract, but the patient was not willing for surgery till her last follow-up at approximately 7 months. The other case (serial # 11 in Table 1) with progression of cataract had visual acuity of 6/12 (0.5) in the operated eye; hence she was not advised surgery for cataract.
Clinically evident epiretinal membrane (ERM) was present in none of the cases of idiopathic and traumatic macular hole group. However, except traumatic macular hole cases, all cases where the specimen could be examined with an electron microscope revealed the presence of ERM. None of the operated patients developed any known complications of vitreous surgery. Only those patients with open macular hole at three months were subjected to resurgery.
In patients with idiopathic macular hole (n = 10), the average age was 56.4 years (range 46-66 years, SD = 7.834). Mean preoperative BCVA was 0.1737 (SD = 0.08). Six patients were in stage III macular hole and four were in stage IV macular hole. Lens was clear in six, cataractous in three and an intraocular lens was present in one patient. Mean duration of onset of visual symptoms was 4.2 months (range 1-12 months, SD = 3.05). Mean size of the macular hole was 500 µm (SD = 81.65). Postoperatively, the holes were closed in all but one patient with myopic macular hole (90% primary surgery success). Mean postoperative BCVA was 0.485 (SD = 0.24). BCVA was 0.5 or better in five (50%) cases.
In myopic macular hole (n = 10), the average age of the patients was 62.1 years (range 48-72 years, SD = 7.046). Mean preoperative BCVA was 0.1347 (SD = 0.09). Five patients were in stage III macular hole and five were in stage IV macular hole. Mean duration of onset of visual symptoms was 6.7 months (range 1-24 months, SD = 6.55). Mean size of the macular hole was 520 µm (SD = 154.92). Mean postoperative BCVA was 0.410 (SD = 0.2). BCVA were 0.5 or better in five (50%) cases.
In traumatic macular hole (n = 10), the average age was 23.3 years (range 8-45 years, SD = 9.911). Mean preoperative BCVA was 0.2541 (SD = 0.13). Eight patients were in stage III macular hole and two were in stage IV macular hole. Mean duration of onset of visual symptoms was 4 months (range 2-7 months, SD = 1.67). Mean size of the macular hole was 550 µm (SD = 143.37). Postoperatively the holes were closed in all patients (100% primary surgery success). Mean postoperative BCVA was 0.577 (SD = 0.3). BCVA was 0.5 or better in six (60%) cases.
In negative controls (n = 10) for electron microscopic features, eight patients had retinal detachment with proliferative vitreoretinopathy and two had tractional retinal detachment as a complication of diabetic retinopathy. The average age was 45.8 years (range 24-66 years, SD = 14.9205).
Electron microscopy features
Twenty-two surgical specimens were chosen to be examined with the electron microscope; however, because of the small size of the tissue samples, seven specimens were lost during washing and dehydration. Therefore, histological observations could be made on only 15 specimens. The case numbers and histopathological features of these specimens are summarised in Table 3. There were fragments of either ILM alone or a complex of ILM and the adherent ERM; no other retinal layers were present. In most of the specimens, the ILM was highly folded. It was identified by its characteristic high electron density, a smooth inner (vitreal) surface that often showed adherent cortical vitreal collagen fibrils, and an outer (retinal) irregular surface (Figure 2c, d), often containing cell organelles of Müller glia.
The characteristic features noted with the surgical specimens in different aetiologies are as follows:
Idiopathic macular hole: In these cases, a complex of the ILM and ERM was present in surgical specimens. The ERM consisted of cortical vitreous and cells that were attached to it. The cellular elements were of two types: one with a round to oval nucleus that possessed little heterochromatin (presumably fibrous astrocytes), and the other with an indented to elongated nucleus showing rich, patchy, heterochromatin (Figure 1a). Both cell types characteristically possessed a large nucleus with a thin rim of perinuclear cytoplasm (Figure 1a). The cytoplasm of the cells were rich in granular endoplasmic reticulum. Figure 1c). The cytoplasmic processes were found to contain intermediate filaments in bundles (Figure 1b, d). Glycogen and numerous vacuoles were present in the cytoplasm of the fibrous astrocytes (Figure 2a), but were lacking in the other form (Figure 2b), which thus could be of fibroblasts. In two patients with posterior subcapsular cataract (case Nos. 3 and 6), the outer side of the ILM showed profiles of elongated cells with patchy nuclear heterochromatin (Figure 2c). However, due to their poor ultrastructural preservation, the details of the cytoplasmic features could not be resolved, thus leaving their identity unknown. Perhaps they were reactive astrocytes which had migrated from the nerve fibre layer.
Myopic macular hole: In these cases, fragments of ILM were present in the surgical specimens. There was an epiretinal membrane adherent to the ILM in all four cases (Figure 2d). The inner side of the ILM contained densely packed native vitreous collagen as well as newly synthesised collagen fibrils (Figure 3b). The cortical vitreous also possessed cellular processes containing bundles of intermediate filaments (Figure 3a). Cell bodies with nucleus devoid of heterochromatin (Figure 3a), presumably of the fibrous astrocytes, were found in the deeper vitreous, as in the idiopathic macular hole cases. In one case, (No. 18), the retinal side of the ILM showed features of proliferative Müller cells. The processes of the Müller cells, containing filamentous structures and smooth endoplasmic reticulum, were aligned in an irregular way (Figure 3c).
Traumatic macular hole: In these cases, fragments of only ILM were present in the surgical specimens. The inner surface of the ILM showed native collagen fibrils that remained adhered to it (Figure 3d).
Negative controls: In all three cases, the ERM was present. It consisted of the cortical vitreous that adhered to the fragment of the ILM. The cortical vitreous contained abundant newly formed, thick collagen fibrils (Figure 4a). Elongated cells, presumably fibrocytes with electron dense cytoplasm were intimately adhered to the cortical vitreous (Figure 4b). Besides, a few cells with cytoplasmic melanin pigment and multivesicular bodies were also found in the same vicinity (Figure 4c). Broad cellular processes were also observed to lie enmeshed in the vitreal collagen fibrils. They contained thin, actin filaments as well as surface microvilli (Figure 4d), suggestive of myofibroblastic origin.
The purpose of this study was to study the electron microscopic features of peeled-ILM and also to describe the visual outcome following surgery. The study also compared the structural differences, if any, between the ILM and the adherent ERM of macular holes with different aetiologies and also with other diseases like proliferative vitreoretinopathy.
In the present series, we have reported closures of the holes in 90 % of cases after primary surgery in 20 cases of idiopathic and myopic macular hole group. Second surgery, which included removal of the large sized ILM, resulted in 100% closure of the holes in both groups. Visual acuity did not improve, however, in two cases; one with idiopathic macular hole, and one with myopic macular hole which required second surgery. In traumatic macular hole group, closure of the macular hole along with improvement in visual acuity was achieved in 100% cases. The overall success in terms of vision improvement of two or more Snellen's lines was noted in 24 out of 30 eyes (80%). No intraoperative or postoperative complication arose apart from failure of closure in two cases. These clinical data commensurate with those of other reported series.19202122
Removal of the ILM is presumed to prevent the formation of ERM in which the former provides the necessary scaffold.23 The ERM is a sheet of cortical vitreous collagen that remains adhered to the ILM and cells of diverse origin. The cells reported to occur in the ERM are fibroblasts, fibrous astrocytes, hyalocytes, retinal pigment epithelium and myofibroblasts.24252627 The fibrous material of the cortical vitreous comprises the collagen fibrils. These are of two types, the native, thin vitreous collagen (about 12-16 nm in diameter) and the newly formed thick collagen fibrils (about 18-22 nm in diameter).
In this study, under the electron microscope, the surgical specimens were observed to be either fragments of ILM alone or a complex of ILM and adherent ERM. In all 15 cases, the ILM was unequivocally identified. The ERM was found in all cases except the three post-traumatic cases. The absence of clinically apparent ERM, while the same being detected on EM, in idiopathic macular hole cases could not be correlated. Myofibroblasts were observed only in negative controls. These cells were identified by the presence of dense aggregates of thin (actin) filaments in their cytoplasmic processes as well as surface microvilli, as reported earlier.27 This study also found pigment-laden cells and fibrocytes in the ERM of control cases. The melanin pigment containing cells could be macrophages, since they possessed multivesicular bodies, a characteristic feature for active macrophages. Ishida et al noted only myofibroblasts in the ERM in cases of long-standing retinal detachment with signs of progression to proliferative vitreoretinopathy.28 Thus several previous reports and results from our study tend to emphasise myofibroblasts as the main contributor to proliferative vitreoretinopathy.2829
Cell proliferation, especially of the myofibroblasts is believed to contribute to tangential traction, which may play an important role in macular hole formation and subsequent retinal detachment.623 Contraction of myofibroblasts may cause enlargement of the macular hole.2427 The newly synthesised collagen fibrils in the ERM around the macular hole may assist in the contraction of myofibroblasts.27 In our study, newly synthesised collagen fibrils were observed in all three control cases (case numbers 34, 36 and 39), which thus might play a role in contraction of the myofibroblasts. It is, therefore, vital to consider as to why these cells were not found in any of the samples from idiopathic and myopic macular hole cases. Yoon et al studied a series of idiopathic macular hole cases and reported a proliferation of myofibroblasts in three cases out of seven stage III hole and in all three cases of stage IV hole.27 According to them, cell proliferation is involved in initiating macular hole formation. However, in this study, although no myofibroblast-like cells were noted in any of the nine cases from the idiopathic and myopic macular hole series, there was evidence of fibroblast differentiation besides fibrous astrocytes, and there was a possibility that these fibroblast cells as well as the fibrous astrocytes may have caused contraction of the hole. As fibroblast cells differentiate into myofibroblasts, it is expected that either fibroblasts or myofibroblasts be present in idiopathic and myopic macular holes.
The ILM is supposed to act as a scaffold for cellular proliferation. A number of clinical reports have shown proliferation of myofibroblasts and fibrous astrocytes on the inner (vitreal) side of the ILM in macular hole formation. Besides, proliferation of Müller cells is reported to occur with the formation of macular hole. Madreperla et al have reported opercula to consist of a thin cortical vitreous on its inner surface and processes of fibrous astrocytes and Müller cells on the outer (retinal) side, being identified unequivocally by their known ultrastructural features.6 They are of the view that Müller cells and fibrous astrocytes do proliferate in an attempt to seal the macular hole, and that their proliferation may not be due to secondary changes occurring after retinal detachment. This study, noted Müller cell proliferation in only one case of the myopic macular hole series, the significance of which is not clear.
This clinical study, found evidence of glial proliferation on the inner side of the ILM in cases of idiopathic and myopic macular holes. There was extensive proliferation of the fibrous astrocytes and their elongated processes were enmeshed in the cortical vitreous. Cell proliferation was apparently higher in stage IV macular hole, because their duration was longer than that of stage III holes. The newly proliferated cells possessed a large nucleus and scanty peripheral cytoplasm, as would be expected. Ishida et al studied myopic macular holes in five Japanese patients and found fibrous astrocytes as the major cell type in ERM.28 The cortical vitreous contained abundant newly synthesised collagen fibrils, and according to them this synthesis might have been regulated by those glia. The collagen of the cortical vitreous may exert tangential traction in the macula, causing retinal detachment in these cases. This was the picture in our study too, with four myopic macular hole cases, showing proliferation of fibrous astrocytes as well as synthesis of new collagen in the ERM.
It is concluded that ILM peeling is a reasonable surgical adjunct to vitrectomy to treat macular holes of idiopathic, myopic and traumatic aetiology. The electron microscopic structure of ILM peel may help in better understanding of aetiopathogenesis of macular holes of different aetiologies.
The electron microscope work was carried out at The Sophisticated Analytical Instrumentation Facility (DST), Department of Anatomy, AIIMS, New Delhi.
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