Internal Limiting Membrane Specimens Research Articles

Premacular Cells as Source of Neurotrophic Factors in Idiopathic Macular Holes

ABSTRACT 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.

Biomechanical Properties of the Internal Limiting Membrane after Intravitreal Ocriplasmin Treatment

Purpose: To assess the stiffness of the human internal limiting membrane (ILM) and evaluate potential changes of mechanical properties following intravitreal ocriplasmin injection for vitreomacular traction. Methods: This is an interventional comparative case series of 12 surgically excised ILM specimens consecutively obtained from 9 eyes of 9 patients after unsuccessful pharmacologic vitreolysis with ocriplasmin. During the same time period, 16 specimens from 13 other eyes without ocriplasmin treatment were harvested during vitrectomy and served as controls. All patients presented with macular holes or vitreomacular traction and underwent vitrectomy with ILM peeling either with or without brilliant blue (BB) staining. All specimens were analyzed using atomic force microscopy with scan regions of 25 × 25 μm. In all specimens, both the retinal side and vitreal side of the ILM were analyzed. Results: Atomic force microscopy revealed no significant differences in elasticity of ILM specimens removed from eyes with or without ocriplasmin treatment. Undulated areas of the retinal side presented stiffer than the vitreal side of the ILM. Topographical mapping of both the vitreal and retinal side of the ILM showed no apparent alteration of the morphology in ocriplasmin-treated eyes compared to untreated eyes. Staining with BB resulted in an increase of tissue stiffness. Conclusions: Intravitreal injection of ocriplasmin does not change biomechanical properties of the human ILM. There is no evidence of a potential enzymatic effect of ocriplasmin interfering with the stiffness of this basement membrane.

Pathology of Internal Limiting Membrane Specimens Following Intravitreal Injection of Ocriplasmin

To describe ultrastructure and immunocytochemistry of internal limiting membrane peelings after unsuccessful treatment with ocriplasmin, to compare with untreated eyes, and to correlate with clinical imaging data. Interventional comparative case series. Internal limiting membrane specimens were removed from 10 eyes with small macular holes and vitreomacular traction during vitrectomy after intravitreal ocriplasmin injection without release of traction or closure of macular holes during follow-up. Based on optical coherence tomography analysis, specimens from 10 other eyes without ocriplasmin treatment served as controls. All specimens were processed as flat mounts for phase-contrast microscopy followed by immunolabeling for fluorescence microscopy and embedding in epoxy resin with serial sectioning for transmission electron microscopy. Despite the absence of contractive epiretinal membranes on optical coherence tomography, we found epiretinal cells and vitreous collagen fibrils on the internal limiting membrane in specimens removed from eyes with and without previous pharmacologic vitreolysis. Immunolabeling revealed glial cells and hyalocytes in macular holes, whereas myofibroblasts were predominant in vitreomacular traction. There was no apparent damage of the vitreoretinal interface after unsuccessful pharmacologic vitreolysis compared to untreated controls. Epiretinal cell proliferation and vitreous collagen fibrils with close adhesions to the internal limiting membrane are not always detectable by optical coherence tomography or may not have been recognized. Since they are associated with unsuccessful ocriplasmin treatment, presence and topography of epiretinal cells and vitreous collagen remnants on the internal limiting membrane should be further elucidated in order to refine criteria and indications for case selection in pharmacologic vitreolysis.

Cells at the vitreoretinal interface in small full-thickness macular holes.

To report on total number, distribution, and type of cells at the vitreomacular interface in small full-thickness macular holes. Internal limiting membrane specimens were removed from 20 consecutive patients with macular holes <250 μm at times when pharmacologic vitreolysis was not available. Specimens were flat mounted and investigated by phase contrast and interference microscopy and immunocytochemistry. Clinical data were documented including optical coherence tomography analysis using the caliper function. Thirteen antibodies were used for glial cells, hyalocytes, macrophages, retinal pigment epithelial cells, different types of collagen, alpha-smooth muscle actin, and proliferating cells. There was a positive correlation between macular hole size and cell density at the internal limiting membrane (Spearman's Rho: r = 0.519, P = 0.019). Mostly, single glial cells were found on the internal limiting membrane. In five patients, cell clusters were present. There was a strong immunoreactivity for glial cell markers. Immunoreactivity of hyalocyte markers, alpha-smooth muscle actin, and Ki-67 was found in cell clusters but otherwise sparse. Single cells of glial origin without signs of proliferation or contraction are present in eyes with small full-thickness macular holes. In some eyes, however, clusters of cells can be seen, capable of proliferation and exerting tangential traction. Our findings emphasize the need for better visualization of the vitreoretinal pathology by optical coherence tomography, especially to distinguish between single cells and cell clusters.

Lamellar Hole–Associated Epiretinal Proliferation in Comparison to Epiretinal Membranes of Macular Pseudoholes

To compare immunocytochemical and ultrastructural characteristics of "lamellar hole-associated epiretinal proliferation" in lamellar macular holes with "conventional epiretinal membrane" in macular pseudoholes. A consecutive observational case series, laboratory investigation. We analyzed surgically excised flat-mounted internal limiting membrane specimens and epiretinal membrane specimens removed from 25 eyes of 25 patients with lamellar macular holes (11 eyes) and macular pseudoholes (14 eyes) using interference and phase-contrast microscopy, immunocytochemistry, and transmission electron microscopy. By spectral-domain optical coherence tomography, epiretinal material of homogenous reflectivity without contractive properties was categorized as lamellar hole-associated epiretinal proliferation, whereas tractional epiretinal membranes presenting contractive properties were termed conventional epiretinal membrane. Lamellar hole-associated epiretinal proliferation was seen in 73% of eyes with lamellar macular hole. Eyes with macular pseudohole presented with conventional epiretinal membrane. In lamellar hole-associated epiretinal proliferation, positive immunoreactivity for anti-glial fibrillary acidic protein, hyalocyte markers, and anti-collagen type I and III was seen. In contrast, specimens of macular pseudoholes were positive for α-smooth muscle actin and anti-glial fibrillary acidic protein, predominantly. Cellular ultrastructure showed that lamellar hole-associated epiretinal proliferation of lamellar macular holes mainly consisted of fibroblasts and hyalocytes, whereas myofibroblasts dominated in conventional epiretinal membranes of macular pseudoholes. Cells within lamellar hole-associated epiretinal proliferation appear to originate from vitreous and possess less contractive properties than cells of conventional epiretinal membranes. Our findings point to differences in pathogenesis in a subgroup of lamellar macular holes presenting lamellar hole-associated epiretinal proliferation on the retinal surface.

Vital dyes increase the rigidity of the internal limiting membrane

To assess the stiffness of the natural human internal limiting membrane (ILM) and evaluate potential changes of the mechanical properties following staining with brilliant blue (BB) and indocyanine green (ICG). Unstained ILM specimens were obtained during ophthalmic surgical procedures. After removal, the specimens were dissected into five parts. Two fragments were stained with BB and ICG, respectively, for 1 min, another two specimens were stained similarly followed by additional subsequent illumination using a standard light source (PENTA LUX x 50, Ophthalmologische Systeme GmbH Fritz Ruck). The fifth part served as an untreated control. All specimens were then analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 × 10 μm were chosen and stiffness was determined by using AFM in a force spectroscopy mode. The force curves were plotted with a data rate of 5000 Hz. In all specimens both the retinal side and vitreal side were analyzed. Staining resulted in a significant increase in tissue stiffness. An increase was seen both for the vitreal (BB: P<0.001; ICG: P<0.01) and retinal side (BB: P<0.01; ICG: P<0.01), with the retinal side being significantly stiffer in all control and stained samples. Additional illumination after staining did further increase tissue rigidity in most samples but not significantly. Staining significantly increases the stiffness of the human ILM. This might explain the fact that the stained ILM can be removed more easily and in larger fragments during vitreoretinal surgical procedures compared with unstained ILM.

EPIRETINAL CELL PROLIFERATION IN MACULAR PUCKER AND VITREOMACULAR TRACTION SYNDROME

To describe new details of epiretinal cell proliferation in flat-mounted internal limiting membrane specimens. 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. 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. 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.

Histopathologic analysis of the internal limiting membrane surgically peeled from eyes with diffuse diabetic macular edema

To investigate the pathogenesis of diffuse diabetic maculae edema (DME). Internal limiting membrane (ILM) specimens were surgically peeled from 16 eyes of 15 patients with diffuse DME (diabetic retinopathy (DR) group) and from 12 eyes of 12 patients without diabetes (non-DR group). The specimens were then examined by light microscopy (LM), transmission electron microscopy (TEM), and immunohistochemistry. Examination by LM revealed numerous cells on the inner surface of the ILM specimens in the DR-group, among which five different cell types (glial cell, fibroblast-like cell, macrophages, neutrophils, and lymphocytes) were confirmed. Examination by TEM revealed that the thickness of the peeled ILMs in the DR group was significantly greater, and four types of cellular element (glial, fibroblast-like, macrophages, and lymphocytes) were confirmed on the surface of the vitreous side of the specimens. The existence of glial cells and macrophages was confirmed by immunohistochemistry. In diffuse DME, the ILM is thickened and a variety of cellular elements, especially numerous kinds of inflammatory cell, adhere to the inner surface of the ILM. To attenuate diffuse DME, vitrectomy combined with ILM peeling to remove the inflammatory cells and the physical barrier might be effective.

RESIDUAL CELLULAR PROLIFERATION ON THE INTERNAL LIMITING MEMBRANE IN MACULAR PUCKER SURGERY

To provide pathology data on the completeness of epiretinal membrane (ERM) removal with and without internal limiting membrane (ILM) peeling. Twenty-two patients with idiopathic ERM formation underwent vitrectomy with ERM removal and subsequent staining of the vitreomacular interface with brilliant blue. If the ILM was still present after ERM removal, it was peeled off. Both ERM and ILM specimens were harvested in different containers and prepared for flat-mount phase-contrast and interference microscopy, immunocytochemistry, and transmission electron microscopy. In 14 patients (64%), the ILM was still present at the macula after ERM removal. On average, 20% (range, 2-51%) of the total cell count was left behind at the ILM if the ERM was removed only. There were mainly glial cells on the ILM, and few hyalocytes. In nine eyes, the cells were forming cell clusters. In 8 patients (36%), both ERM and ILM were removed together. Electron microscopy showed cellular proliferation directly attached to the ILM in these eyes, whereas in the sequentially peeled group, there was collagen interposed between the ERM and the ILM. Surgical ERM removal resulted in splitting of the vitreous cortex in these eyes, leaving the ILM with residual cells behind. Simple ERM removal results in sufficient separation of fibrocellular tissue in one third of cases, only. In 2 of 3 patients with idiopathic ERM, the vitreous cortex splits when the ERM is removed, leaving an average of 20% of the total cell count behind on the ILM. As these cells are capable of proliferation and causing ERM recurrence, staining of the ILM with subsequent removal seems beneficial in macular pucker surgery.

Histopathology of Internal Limiting Membrane Peeling In Traction Induced Maculopathies

Purpose: To compare the presence of cell fragments and entire cell bodies on the retinal side of the internal limiting membrane (ILM) after removal with and without dye assistance in traction maculopathies. Methods: En-bloc removal of the ILM and epimacular tissue was performed during vitrectomy in 75 eyes without dye-assistance and in 45 eyes with assistance of either Brilliant Blue G (BBG) or Trypan Blue (TB). We included 79 eyes with macular holes (MH) and 41 eyes with other traction maculopathies. All ILM specimens were processed by serial sectioning preparation for light microscopy. Exclusively, if cellular elements of more than 2 μm in diameter were found on the retinal side of the ILM by light microscopy, specimens were prepared for transmission electron microscopy. Results: 23 (19%) specimens of this series demonstrated such cell fragments and entire cell bodies on the retinal side of the ILM. Specimens removed from MH eyes demonstrated less frequently retinal cell debris than specimens removed from other traction maculopathies. If epiretinal cell proliferation was seen, cellular debris on the ILM was significantly more frequent, irrespective of BBG or TB assistance. Conclusions: Removal of cellular structures on the retinal side of the ILM during ILM peeling appears to be associated with epiretinal cell proliferation. The presence of cell fragments and entire cell bodies on the retinal side of the ILM seems unrelated to the use of BBG and TB. Epiretinal membranes with wrinkling and folding of the ILM may contribute to structural changes that facilitate pulling off parts of cells or entire cell bodies during ILM peeling.

Staining of the Internal Limiting Membrane with the Use of Heavy Brilliant Blue G

Background: Brilliant blue G (BBG) is frequently used in chromovitrectomy to facilitate internal limiting membrane (ILM) peeling. A study was initiated to evaluate if heavy BBG is safe and effective in staining the ILM. Methods: We studied 30 eyes, 23 with idiopathic macular holes and 7 of patients with diabetic macular edema. Removal of the ILMs was assisted by heavy BBG staining. In cases with histopathological correlation the ILMs were evaluated with hematoxylin and eosin, Masson’s trichrome, periodic acid-Schiff and glial fibrillary acidic protein staining. In addition, immunohistochemistry was also performed using specific antibodies for vimentin, neuron-specific enolase, factor VIII and CD68. Using the Image-Pro Plus software of Media Cybernetics Co. we found an average thickness in ILMs. Results: Of the ILM specimens sent, 19/30 (63.33%) could not be processed properly because of the limited sample material, recognizing only fragments of dispersed fibrillar material. In macular hole ILMs we found an average thickness of 1.3 ± 0.65 µm, and in diabetic macular edema ILMs an average thickness of 6.2 ± 1.4 µm. Conclusions: In heavy BBG-assisted ILM peeling we observed no intraoperative or postoperative complications after a mean follow-up of 12 months. Heavy BBG could be an effective and safe vehicle for staining the ILM.

Immunocytochemical and Ultrastructural Evidence of Glial Cells and Hyalocytes in Internal Limiting Membrane Specimens of Idiopathic Macular Holes

To provide new information on epiretinal cell proliferation and the cells' origin in idiopathic macular holes and to overcome the effects of embedding and sectioning preparation procedures on cell-distribution patterns. Interference and phase-contrast microscopy, immunocytochemistry, and scanning and transmission electron microscopy were performed on surgically excised whole-mounted internal limiting membrane (ILM) specimens removed from 60 eyes with idiopathic macular holes. Cell distribution and cell morphology were correlated with immunocytochemical staining characteristics. Twelve cell type-specific antibodies were used to detect glial cells, hyalocytes, retinal pigment epithelial cells, retinal ganglion cells, and immune cells. Cell viability was analyzed. Epiretinal cell proliferation was found in all ILM specimens, irrespective of the stage of the macular hole. Cell density showed a broad variety. Immunocytochemistry frequently revealed simultaneous expression of GFAP/CD45, GFAP/CD64, GFAP/CD68, GFAP/CRALBP, and GFAP/CD90. Some cells presented with intracellular contractile filaments (anti-αSMA); others were not immunoreactive to any antibody examined. The percentage of viable cells showed a broad variety with a mean of 73% (SD 29%). Electron microscopy demonstrated glial cells, hyalocytes, and myofibroblast-like cells. The presence of epiretinal cells at the ILM in all macular hole stages strongly suggests a substantial involvement of cell migration and proliferation in the course of macular hole development. Glial cells and hyalocytes play the predominant role in epiretinal cell proliferation. Given the co-expression of glial cell and hyalocyte markers, transdifferentiation of epiretinal cells needs further elucidation, especially with respect to αSMA-positive cells leading to traction at the vitreoretinal interface.

Macular function and ultrastructure of the internal limiting membrane removed during surgery for idiopathic epiretinal membrane

To determine the effect on macular function of removal of the internal limiting membrane (ILM) during epiretinal membrane surgery. Prospective, interventional study. Twenty-two eyes of 22 patients with idiopathic epiretinal membranes undergoing surgery. Histologic analysis of the ILM specimens were classified based on amount of retinal tissue fragments attached to the removed ILM. The ophthalmic examinations were assessed prospectively. The visual acuity, optical coherence tomography, and multifocal electroretinography at baseline, 1 and 3 months after surgery. The visual acuity at baseline, 1 and 3 months after surgery were not different between the small and large retinal debris groups. On multifocal electroretinography, there was no significant difference in response of the N1 amplitude, N1 latency, and P1 amplitude between the small and large retinal debris groups at baseline, and 1 and 3 months after surgery. However, compared to the small debris group, the P1 latencies in the foveal areas were delayed in the large debris group at 1 and 3 months (P = 0.020, P = 0.047, respectively). The central retinal thickness was significantly reduced in the large debris group, while the status of the photoreceptor line was not different based on optical coherence tomography between the small and large debris groups 3 months after surgery (P = 0.047). Large amounts of retinal debris attached to the removed ILM did not affect visual acuity. However, the ultrastructural findings of Muller cell damage might be related to subtle macular dysfunction on multifocal electroretinography after surgery.

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

PurposeTo 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...

Does the Presence of an Epiretinal Membrane Alter the Cleavage Plane during Internal Limiting Membrane Peeling?

To determine whether the presence of a clinically and/or microscopically detectable epiretinal membrane (ERM) alters the cleavage plane during internal limiting membrane (ILM) peeling. Retrospective, observational, immunohistochemical study of ILM specimens using archival formalin-fixed, paraffin-embedded tissue. Fifty-one patients who had had ILM excision. Fifty-one ILM specimens peeled during vitrectomy for various etiologies were examined by light microscopy. The removal of ILM was assisted using Trypan blue (n = 30), indocyanine green (n = 7), or brilliant blue G (n = 14). Monoclonal antibodies to glial fibrillary acidic protein and to neurofilament protein were used to detect glial or neuronal cells respectively on the vitreous or retinal surfaces of the ILM. Specimens were divided into 2 groups: ILM peeled for full-thickness macular hole (MH; n = 31) and ILM peeled after removal of clinically detectable ERM (n = 20). Primary outcome measure was the localization of immunohistochemical markers to neuronal or glial cells on the vitreous or retinal surfaces of ILM. The secondary outcome measure was the correlation of the results of the primary measure with the dyes used to facilitate ILM peeling. Glial and/or neuronal cells were detected on the retinal surface of the ILM in 10 of 31 (32%) of the MH ILM specimens and in 13 of 20 (65%) of the ILM peeled after ERM excision; the difference was significant (P = 0.02). There was no association between the presence of neuronal and glial cells with the type of dye used (P = 0.2). Of the 23 ILM specimens with cells attached to the retinal surface, 21 (91%) were associated with clinical and/or histologic evidence of ERM and 2 (9%) were not. The correlation between the presence of cells on the vitreous and the retinal surfaces of ILM was high (P<0.0001). The findings suggest that ERM may be associated with sub-ILM changes that alter the plane of separation during ILM peeling. This study does not confirm any influence of dyes on the cleavage plane during surgery.

Histological observationsof the macular epiretinal membranes from pathological myopic eyes with macular hole

：Objective To investigatethe histological changes of the internal limiting membrane (ILM) in pathological myopiceyes, and the relationships between those changes and the formation of macular hole.Methods The clinical data of 34 patients (34 eyes)with pathological myopia and maeularhole, who had undergone vitrectomy were retrospectively analyzed. All the patients had adiopter over--6.00 D, their ocular axial length ranged from 26.00 to 33.12 mm with anaverage of 27.74 mm. There were 5 eyes without retinal detachment (macular holegroup)while the other 29 eyes all had posterior retinal detachment (RD group). A standardthree-port pars plana vitrectomy was performed in all eyes. The existence of Weiss ringwas considered as the sign of posterior vitreous detachment. The epiretinal membranes from34 eyes and ILM specimens from 19 eyes were stained by hematoxylin-eosin (HE) and acetateurany[ acetate-lead citrate, and then investigated under optical microscope andtransmission electron microscope. Results The Weiss ring was observed in 5 eyes during thesurgery, multilayer of vitreous tissue resides on the retinal surface in 24 eyes. Theresults of optical microscope showed that the epiretinal membranes were composed ofvitreous collagen, astrocytes and extracellular matrixes. The results from transmissionelectron microscope showed a sandwich structure (ILM-vitreous collagen-cells) in 5 eyes,and ILM damage, surface traction and astrocytes migration in 1 eye. Conclusions Splittingof posterior surface of vitreous and surface structure change of ILM are the majormechanisms for the formation and progression of macular holes, and even retinal detachmentin high myopic eyes. Key words: Myopia; degenerative/complications; Retinal perforations/etiology; Epiretinal membrane/pathology

Internal limiting membrane surrounding idiopathic stage 4 macular hole contains bundles of actin microfilaments

Abstract Purpose To study internal limiting membrane (ILM) specimens obtained from a case of stage 4 macular hole surgery with confocal microscopy and investigate the presence of actin microfilaments. Methods A 68 year‐old woman underwent 20‐gauge transconjunctival sutureless vitrectomy for stage 4 idiopathic macular hole. Triamcinolone –acetonide was used for visualizing the vitreous hyaloid. Following vitrectomy, the ILM was stained with Brilliant blue dye, then peeled off using an intraocular forceps. Two separate ILM specimens were obtained during surgery; the first around the macular hole and the second one disc diameter away from the macular hole up to the temporal vascular arcades and the optic disk. At the end of the surgery, air‐gas (C3F8 16%) exchange was performed. The ILM specimens were studied immunohistochemically using confocal microscopy after labeling with antibodies to actin and fibronectin. Results The ILM specimen around the macular hole was stained positive for both fibronectin and actin, whereas the ILM specimen away from the macular hole was stained positive only for fibronectin. Preoperative evaluation of the macular hole biomicroscopically and imaging with optical coherence tomography showed absence of any epiretinal membrane (ERM). Conclusion ILM surrounding stage 4 macular hole contains bundles of actin microfilaments, suggesting the presence of cells with contractile properties even in the absence of associated ERM. These findings suggest that tangential traction may contribute to the pathogenesis of late stage macular hole development and/or enlargement and support removal of ILM in late stage macular hole surgery.

Blood-Assisted Internal Limiting Membrane Peeling for Macular Hole Repair

To evaluate the efficacy of autologous heparinized whole blood in assisting internal limiting membrane (ILM) peeling by coating the ILM for macular hole (MH) repair. Prospective, interventional case series. Twenty-nine patients (32 eyes) who underwent blood-assisted ILM peeling for MH repair. Patients in whom stage 2-4 idiopathic MHs had developed and who desired surgery were enrolled in this study. After core vitrectomy, autologous heparinized whole blood was applied to cover the macula and to coat the surface of the macular area in the fluid-filled vitreous cavity. The redundant blood was removed and only a very thin film of blood was left on the macular area. The blood-coated ILM was removed by forceps in a circular fashion. To confirm the removed membrane was the ILM, the first 10 specimens were examined by electron microscopy (EM). The MH closure rate, the interval mean visual acuity (before and after surgery), retinal changes, and the EM results of the ILM specimens. All 32 eyes in 29 patients completed 12 months of follow-up. The ILM were coated by autologous heparinized whole blood, removed without difficulty, and confirmed by EM. The whole blood highlighted the contrast of the coated and noncoated areas during the ILM peeling procedure. The MHs were closed in all surgical eyes with a single surgery (100%). Compared with study entry, the mean logMAR best-corrected visual acuity 12 months after surgery improved significantly (1.02 and 0.53, respectively; P<0.001). At 12 months of follow-up, 31 eyes (96.9%) had stable or improved vision. No toxic fundus changes were observed during follow-up. Autologous heparinized whole blood coated the ILM and facilitated visibility during ILM peeling. Autologous heparinized whole blood is a cost-effective and useful tool for assisting MH surgery.

VITAL DYES FOR MACULAR SURGERY

To analyze and compare the ultrastructure of the retinal cleavage plane of the internal limiting membrane (ILM) removed during dye-assisted macular surgery. 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. 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. 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.

ANALYSIS OF THE NEURONAL MARKER PROTEIN GENE PRODUCT 9.5 IN INTERNAL LIMITING MEMBRANES AFTER INDOCYANINE-GREEN ASSISTED PEELING

Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.