Vitreal Side Research Articles

CLINICOPATHOLOGIC CHANGES OF VITREOMACULAR INTERFACE IN IDIOPATHIC EPIRETINAL MEMBRANE WITH DISORGANIZATION OF RETINAL INNER LAYERS.

To compare the pathological characteristics of the vitreomacular interface of the idiopathic epiretinal membrane with and without disorganization of retinal inner layers (DRIL) and to correlate with clinical data. In this clinicopathologic study, the samples of epiretinal membrane and internal limiting membrane were extracted from DRIL(+) (19 eyes) and DRIL(-) (22 eyes) idiopathic epiretinal membrane eyes. Ultrathin series sectioning for transmission electron microscopy was observed and correlated with surgery status and prognosis. All idiopathic epiretinal membrane eyes presented fibrocellular membranes accompanied by vitreous collagen, glial cells, and myofibroblasts, regardless of association with DRIL. A robust signal indicative of Collagen Type VI was observed in eyes DRIL(-), whereas Collagen Type I was discovered in DRIL eyes. Cell debris and microvascular basement membrane were seen on the retinal side of DRIL eyes and a larger cell count on the vitreous side. These have more intraoperative complications and less surgery benefit. Although internal limiting membrane peeling seems important, the histopathologic findings underscore the potential for retinal injury in DRIL(+) idiopathic epiretinal membrane eyes. This suggests that further research is needed to investigate individual preoperative assessment and to modify surgical procedures.

Retinal Development in a Precocial Bird Species, the Quail (Coturnix coturnix, Linnaeus 1758).

The quail (Coturnix coturnix, Linnaeus 1758), a notable model used in developmental biology, is a precocial bird species in which the processes of retinal cell differentiation and retinal histogenesis have been poorly studied. The purpose of the present research is to examine the retinogenesis in this bird species immunohistochemically and compare the results with those from previous studies in precocial and altricial birds. We found that the first PCNA-negative nuclei are detected at Stage (St) 21 in the vitreal region of the neuroblastic layer, coinciding topographically with the first αTubAc-/Tuj1-/Isl1-immunoreactive differentiating ganglion cells. At St28, the first Prox1-immunoreactive nuclei can be distinguished in the vitreal side of the neuroblastic layer (NbL), but also the first visinin-immunoreactive photoreceptors in the scleral surface. The inner plexiform layer (IPL) emerges at St32, and the outer plexiform layer (OPL) becomes visible at St35-the stage in which the first GS-immunoreactive Müller cells are distinguishable. Newly hatched animals show a well-developed stratified retina in which the PCNA-and pHisH3-immunoreactivies are absent. Therefore, retinal cell differentiation in the quail progresses in the stereotyped order conserved among vertebrates, in which ganglion cells initially appear and are followed by amacrine cells, horizontal cells, and photoreceptors. Müller glia are one of the last cell types to be born. Plexiform layers emerge following a vitreal-to-scleral gradient. Finally, our results suggest that there are no significant differences in the timing of different events involved in retinal maturation between the quail and the chicken, but the same events are delayed in an altricial bird species.

An analysis of the correlation between diabetic retinopathy and preretinal oxygen tension using three-dimensional spoiled gradient-recalled echo sequence imaging

BackgroundThe aims of this study were to evaluate the levels of preretinal oxygen tension in patients with diabetes who did not have hypertension by using three-dimensional spoiled gradient-recalled (3D-SPGR) echo sequence imaging and to explore the correlation between diabetic retinopathy (DR) and changes in preretinal oxygen tension.MethodThis study involved 15 patients with type 2 diabetes without hypertension, who were divided into a diabetic retinopathy (DR) group (n = 10 eyes) and a diabetic non-retinopathy (NDR) group (n = 20 eyes), according to the results of a fundus photography test. Another healthy control group (n = 14 eyes) also participated in the study. The preretinal vitreous optic disc area, nasal side, and temporal side signal intensity of the eyes was assessed before and after oxygen inhalation with the use of 3D-SPGR echo magnetic resonance imaging (MRI). The signal acquisition time was 10, 20, 30, 40, and 50 min after oxygen inhalation.ResultsThe results showed that, in the DR and NDR groups, the preretinal vitreous oxygen tension increased rapidly at 10 min after oxygen inhalation and peaked at 30–40 min, and the increased slope of the DR group was higher than that of the NDR group. The oxygen tension of the preretinal vitreous gradually increased after oxygen inhalation, and the difference between the DR and NDR groups and the control group was statistically significant (P < 0.05). The preretinal vitreous oxygen tension was higher in the optic disc, temporal side, and nasal side in the NDR group than in the control group, and the difference was statistically significant (P < 0.05). The maximum slope ratios of the optic disc and the temporal side of the DR group were greater than those of the control group, and the difference was statistically significant (P < 0.05).ConclusionThree-dimensional-SPGR echo MRI sequencing technology is useful for detecting preretinal oxygen tension levels in patients with diabetes. It can be used as one of the functional and imaging observation indicators for the early diagnosis of DR.

Cell composition at the vitreomacular interface in traumatic macular holes

PurposeTo 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.MethodsFor 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.ResultsSurgically 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.ConclusionThe vitreomacular interface in TMH and IMH shows significant differences. In TMH, glial cells are a rare finding on the vitreal side of the ILM.

The extracellular matrix complexity of idiopathic epiretinal membranes and the bilaminar arrangement of the associated internal limiting membrane in the posterior retina

PurposeTo study the composition of the internal limiting membrane (ILM) of the retina, the extracellular matrix (ECM) of idiopathic epiretinal membranes (iERMs), and the relationships occurring between the two membranes.MethodsForty-six iERMs, 24 of them associated with the ILM, were collected and included in this study. The investigation has been carried out by immunofluorescence and confocal microscopy on glutaraldehyde- and osmium-fixed epon-embedded samples and on frozen samples. Sections were double or triple labelled with antibodies against vimentin; collagens I, III, IV, α5(IV), and VI; laminin 1 + 2; laminin α2-, α4-, α5-, β1-, β2-, β3-, γ1-, and γ2-chains; entactin; and fibronectin.ResultsiERM thickness was not uniform. Almost 14% of iERMs showed thickenings due to folding of their ECM component under the cell layer. The vitreal side of iERMs was often shorter than the attached ILM. In this case, the ILM resulted folded under the iERM. ILMs contained laminin 111; laminin α2-, α5-, β1-, β2-, and γ1-chains; entactin; collagens I; α5(IV); [α1(IV)]2α2(IV); and VI. Laminins, entactin, and α5(IV) were gathered on the retinal half of the ILM, whereas collagens [α1(IV)]2α2(IV) and I were restricted to the vitreal side. Collagen VI was detected on both sides of the ILM. iERMs expressed laminin 111, collagens III, [α1(IV)]2α2(IV) and VI, entactin, and fibronectin. Entactin co-localized with laminins and collagen IV.ConclusionsAnalysis of laminins and collagen chain expression indicates that ILM contains laminin 111 (former laminin 1), laminin 521 (former laminin 11), laminin 211 (former laminin 2), collagen [α1(IV)]2α2(IV), and collagen α3(IV)α4(IV)α5. In contrast, iERMs express only collagen [α1(IV)]2α2(IV) and laminin 111. In addition, both iERMs and ILMs contain entactin. The presence of three major constituents of the basement membranes co-localized together in iERMs is suggestive for a deranged process of basement membrane formation which fails to assemble properly. In view of the many interactions occurring among its proteins, the ECM of either the iERMs or the ILMs can account for their reciprocal adhesiveness. In addition, the peculiar deposition of the ECM observed in some samples of iERM is suggestive for its involvement in the formation of macular puckers.

Effect of Decorin and Bevacizumab on oxygen-induced retinopathy in rat models: A comparative study.

Purpose:The aims of this study were to evaluate the effects of decorin (DCN) in rat oxygen-induced retinopathy (OIR) model and to compare the results with those of bevacizumab.Methods:Twenty-eight newborn Sprague–Dawley rats were randomly divided into four groups. Group I (control): normoxia plus intraperitoneal (ip) normal saline (NS), Group II (sham): OIR plus ip NS, Group III (DCN): OIR plus ip 0.1 mg/kg DCN, and Group IV (bevacizumab): OIR plus ip 2.5 mg/kg bevacizumab. The OIR model was induced by cycling the oxygen concentration between 50% and 10% every 24 h for 14 days following their birth. In all groups, injections were administered on postnatal day (PD) 15. All animals were sacrificed and their right eyes were enucleated on PD 18. The nuclei of neovascular endothelial cells on the vitreal side of the inner limiting membrane were counted, and vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF)-α immunoreactivity were detected in histopathological and immunohistochemical examinations. One-way analysis of variance and post hoc Tukey tests were used for statistical analyses of the data.Results:In Groups II, III, and IV, the mean neovascular cell nuclei counts were 13.14 ± 1.34, 6.57 ± 1.51, and 6.71 ± 1.49, respectively. The mean neovascular cell nuclei count was significantly reduced in treatment groups compared with sham group (P < 0.001). In immunohistochemical staining, the immunoreactivity of VEGF was 0.07 ± 0.02, 0.97 ± 0.21, 0.37 ± 0.12, and 0.23 ± 0.17, respectively. Likewise, immunoreactivity of TNF-α was 0.02 ± 0.02, 1.11 ± 0.36, 0.37 ± 0.13, and 0.62 ± 0.21, respectively. VEGF and TNF-α immunoreactivity increased markedly in the sham group compared with those in the control group (P < 0.001). VEGF and TNF-α immunoreactivity of treatment groups decreased significantly compared to sham group (P < 0.001).Conclusion:The beneficial effects obtained by DCN administration in OIR model were comparable to the effects of bevacizumab.

A time-dependent study of nano-mechanical and ultrastructural properties of internal limiting membrane under ocriplasmin treatment

Vitreomacular traction (VMT) syndrome has only been surgically treated for a long time. Recently, enzymatic vitreolysis with ocriplasmin has emerged as a possible option to release VMT and, in some cases, close full thickness macular holes (FTMHs). Despite its clinical relevance, gathering information about the ocriplasmin-induced alterations of the Inner Limiting Membrane (ILM) of the retina in a clinical study is a complex task, mainly because of the inter-individual variability among patients. To obtain more insights into the mechanism underlying the drug action, we studied in-vitro the mechanical and morphological changes of the ILM using Atomic Force Microscopy (AFM). To this aim, we measured the ILM average Young's modulus (YM), hysteresis (H) and adhesion work (A) over time under ocriplasmin treatment. Our data unveil a time-dependent increase in the membrane YM of 19% of its initial value, along with changes in its adhesive and dissipative behavior. Such modifications well correlate with the morphological alterations detected in the AFM imaging mode. Taken all together, the results here presented provide more insights into the mechanism underlying the ocriplasmin action in-vivo, suggesting that it is only able to alter the top-most layer of the vitreal side of the membrane, not compromising the inner ILM structure.

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.

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

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. Clinicopathologic study. 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. 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. 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.

Non-viral delivery of chemically modified mRNA to the retina: Subretinal versus intravitreal administration

mRNA therapeutics have recently experienced a new wave of interest, mainly due to the discovery that chemical modifications to mRNA's molecular structure could drastically reduce its inherent immunogenicity and perceived instability. On this basis, we aimed to explore the potential of chemically stabilized mRNA for ocular applications. More specifically, we investigated the behavior of mRNA-loaded lipid-based carriers in human retinal cells (in vitro), in bovine retinal explants (ex vivo) and in mouse retinas (in vivo). We demonstrate a clear superiority of mRNA over pDNA to induce protein expression in different retinal cell types, which was further enhanced by chemical modification of the mRNA, providing up to ~1800-fold higher reporter gene expression compared to pDNA. Moreover, transgene expression could be detected for at least 20 days after a single administration of chemically modified mRNA in vitro. We furthermore determined the localization and extent of mRNA expression depending on the administration route. After subretinal (SR) administration, mRNA expression was observed in vivo and ex vivo. By contrast, intravitreal (IVT) administration resulted in limited expression in vivo. Using ex vivo bovine explants with an intact vitreoretinal (VR) interface we could attribute this to the inner limiting membrane (ILM), which presents a large barrier for non-viral delivery of mRNA, trapping mRNA complexes at the vitreal side. When the vitreous was removed, which compromises the ILM, mRNA expression was apparent and seemed to colocalize with Müller cells or photoreceptors after respectively IVT or SR administration. Taken together, this study represents a first step towards mRNA-mediated therapy for retinal diseases.

Effect of intravitreal and intraperitoneal cyanidin-3-glucoside injection in oxygen-induced retinopathy mouse model.

Purpose:To evaluate the effect of cyanidin-3-glucoside (C3G) in oxygen-induced retinopathy (OIR) mouse model.Methods:In this experimental study, 10 C57BL / 6J type mice exposed to room air comprised two control groups (n = 5 each; a negative control and a group receiving intravitreal sterile dimethyl sulfoxide [IVS DMSO]). Thirty C57BL / 6J type mice exposed to 75% ± 2% oxygen from postnatal day 7 to postnatal day 12 comprised the OIR groups. On postnatal day 12, these mice were randomized into six groups (n = 5 each): two OIR control groups (negative control and IVS DMSO), two intravitreal C3G groups (300 and 600 ng/μL), and two intraperitoneal C3G groups (0.05 and 0.1 mg/kg). We quantified neovascularization by counting endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina and examined histological and ultrastructural changes via light and electron microscopy and apoptosis by terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling.Results:The intravitreal C3G groups yielded lower endothelial cell counts compared with the intravitreal DMSO group. The intraperitoneal high-dose group had lower cell counts compared with the OIR control groups. Electron microscopy revealed significantly less mitochondrial dysmorphology in intravitreal groups and the high-dose intraperitoneal mice. We noted no difference in apoptotic cell count between the controls, low-dose intravitreal, and both intraperitoneal groups. However, apoptotic cell count was significantly higher in the high-dose intravitreal group.Conclusion:C3G suppresses endothelial cell proliferation in an OIR mouse model, leads to a reduced hyperoxia-induced mitochondrial dysmorphology, but increases apoptotic cell death in high concentrations.

Morphologic, Biomechanical, and Compositional Features of the Internal Limiting Membrane in Pathologic Myopic Foveoschisis.

To investigate alterations in the morphologic, compositional, and biomechanical properties of the internal limiting membrane (ILM) in pathologic myopic foveoschisis (MF) eyes. ILM specimens were peeled from 61 eyes with MF and 56 eyes with stage III/IV idiopathic macular hole (IMH) as a control. Samples were analyzed for transmission electron microscopy (TEM), scanning electron microscopy, immunofluorescence, Western blotting, and atomic force microscopy. ILM characteristics were compared between the two groups. TEM findings revealed that thickness of the MF ILMs significantly decreased compared with that of IMH ILMs (0.753 ± 0.215 vs. 1.894 ± 0.247 μm; P < 0.0001). The vitreal side stiffness of the MF ILMs was markedly higher than that of the IMH ILMs (3.520 ± 0.803 vs. 0.879 ± 0.230 MPa, P < 0.0001). Comparing with the IMH group, collagen IV exhibited decreased concentration and different immunofluorescence distribution in ILMs of MF eyes, so also protein α3 (IV), α4 (IV), and α5 (IV). The immunofluorescence staining results showed that astrocytes were observed in none of the IMH eyes and in 12 of 16 MF eyes (75%, P < 0.0001). These alterations in the MF ILMs appear to be associated with Müller cell and astrocyte reactive gliosis. The present findings contribute to a more in-depth understanding of the pathogenesis of MF.

Clinicopathological features of epiretinal membranes in eyes filled with silicone oil.

PurposeThe aim of this case series was to clarify the clinicopathological features of epiretinal membranes (ERMs) that developed in eyes after silicone oil (SO) tamponade to treat rhegmatog-enous retinal detachments (RRDs).Patients and methodsIn the Department of Ophthalmology, Saitama Medical Center, Jichi Medical University, patients with idiopathic ERMs (23 eyes) and ERMs in eyes filled with SO (SO ERMs) after vitreous surgery to treat RRDs (nine eyes) were enrolled from July 2012 to March 2014. ERM tissues obtained intraoperatively were examined histopathologically. Besides the main outcome measure of the pathological findings of the ERM tissues, other outcome measures included the preoperative findings on optical coherence tomography (OCT) images and the surgical findings.ResultsEight (89%) of nine eyes with SO ERMs had bilayered membranes composed of a firm layer on the retinal side with glial cells and extracellular matrix and a fragile sponge-like layer on the vitreous side. The sponge-like layer was composed of emulsified SO surrounded by macrophages. Quantitative analysis showed that the areas with cluster of differentiation 68 (CD68)-positive macrophages identified by immunohistochemistry in eyes with SO ERMs were significantly (P<0.001) larger than those in eyes with idiopathic ERMs. The findings on OCT images were consistent with the pathological features of the SO ERMs. Surgical removal of the SO ERMs was difficult because the sponge-like layer was fragile, and the underlying retina was also fragile due to inflammation.ConclusionSO ERMs are bilayered membranes. Long-standing emulsified SO formed a sponge-like layer and SO (foreign body)-induced granulation and caused retinal inflammation in these eyes, making surgical removal difficult. A preoperative OCT examination is necessary to identify SO ERMs.

Efficacy of Intravitreal injection of 2-Methoxyestradiol in regression of neovascularization of a retinopathy of prematurity rat model

BackgroundRetinopathy of prematurity (ROP) is one of the targets for early detection and treatment to prevent childhood blindness in world health organization programs. The purpose of study was to evaluate the efficacy of intravitreal injection of 2-Methoxyestradiol (2-ME) nanoemulsion in regressing neovascularization of a ROP rat model.MethodsA prospective comparative case - control animal study conducted on 56 eyes of 28 healthy new born Sprague Dawley male albino rat. ROP was induced in 21 rats then two concentrations of 2-ME nanoparticles were injected in right eyes of 14 rats (low dose; study group I, high dose; study group II). A blank nanoemulsion was injected in the right eyes of seven rats (control positive group I). No injections performed in contralateral left eyes (control positive group II). Seven rats (14 eyes) were kept in room air (control negative group). On postnatal day 17, eyeballs were enucleated. Histological structure of the retina was examined using Hematoxylin and eosin staining. Vascular endothelial growth factor (VEGF) and glial fibrillary acidic protein (GFAP) expressions were detected by immunohistochemical studies.ResultsIntravitreal injection of 2-ME (in the two concentrations) caused marked regression of the new vascular tufts on the vitreal side with normal organization and thickness of the retina especially in study group II, which also show negative VEGF immunoreaction. Positive GFAP expression was detected in the control positive groups and study group (I).ConclusionIntravitreal injection of 2-Methoxyestradiol nanoemulsion is a promising effective method in reduction of neovascularization of a ROP rat model.

A preliminary report on the characterization of epiretinal membranes excised from patients affected by macular pucker

Among the various pathologies of the vitreoretinal interface, idiopathic macular pucker (MP) is one of the most puzzling. MPs are characterized by the formation of an epiretinal membrane (ERM) that grows in front of the fovea and results in a major impairment of vision. MPs are also frequently complicated by the deformation of the regular macular anatomy, with stretching and deformation of all retinal layers and loss of the foveal pit. This result is usually referred to the presence of myofibroblast-like cells on the retinal surface that alter macular anatomy possibly exerting a significant traction on ERMs. In order to shed light on the physiopathological events that lead to the development of idiopathic MPs, we carried out an immunofluorescence study by confocal microscopy on ERMs excised from 32 eyes with diagnosis of MP using a panel of antibodies including anti-collagen I, anti-collagen IV, anti-collagen VI, anti-smooth muscle actin (SMA), anti-glial fibrillary acidic protein (GFAP) and anti-vimentin antibodies. Some samples were also challenged with anti-heat shock protein (HSP) 47, anti-HSP 90 and anti-receptor II of the transforming growth factor (TGFβRII) antibodies. ERMs broadly varied in thickness. Mostly, they were formed by a layer of collagen 1 adjacent the internal limiting membrane, collagen IV and a layer of vimentin+ cells. Cells also co-expressed SMA or GFAP. Collagen VI, in contrast, was almost always scanty, frequently within the intracytoplasmic vesicles. Some membranes showed a very high content of collagen IV, so abundant to resemble the distribution of interstitial collagens. Cells were almost always restricted to the vitreal side of the membrane; only rarely they could be seen embedded between layers of extracellular matrix. They were frequently HSP90+ and sometimes they contained collagen-immunoreactive materials in their cytoplasm. They also showed TGFβRII within intracytoplasmic vesicles. All in all, ERMs have many features resembling those characterizing fibrotic processes.

Does Internal Limiting Membrane Peeling Size Matter?

Purpose: A variety of retinal topographical changes occur after internal limiting membrane (ILM) peeling for macular holes including a movement of the fovea toward the optic nerve. This study was carried out to assess the effect of the extent of ILM-peeled area on these changes and postoperative visual acuity. Methods: Prospective single-center study of a consecutive series of patients undergoing macular hole surgery. Preoperative and postoperative optical coherence tomography images were used to assess a variety of measures of retinal morphology. Transmission electron microscopy of the peeled ILM was used to assess residual retinal and vitreous side debris. The area of the ILM peeled was calculated from intraoperative images. Results: Fifty-six eyes of 56 patients were included. The mean area of ILM peeled was 9.5 mm2 (2.4-28.3 mm2). The mean disc-to-fovea distance (DFD) preoperatively was 3703 μm with a mean reduction of 52 μm postoperatively, representing a change of −1.29% with a wide range of −7.04% to 1.36%. Using stepwise linear regression, ILM-peeled area was significantly associated with a change in DFD ( P &lt; .001), extent of a dissociated optic nerve fiber layer appearance ( P &lt; .001), and postoperative visual acuity ( P = .025). Nasotemporal retinal thickness asymmetry was associated with the minimum linear diameter ( P &lt; .001). Conclusion: The ILM-peeled area has a significant effect on changes in retinal topography and postoperative visual acuity separate from macular hole size. Further study is needed to assess the effect of ILM peeled size on visual function and to guide clinical practice.

Intravitreal mobility of nanoparticles: how to make a move toward successful ocular gene delivery?

SummaryIntravitreal injection is an interesting delivery route for the administration of nanoparticles for treatment of a variety of retinopathies. To reach the retina, nanoparticles need to diffuse through the vitreous and travel across the vitreoretinal interface. We have recently optimized an ex vivo assay that allows to measure the mobility of fluorescent nanoparticles in intact vitreous humour on a single‐particle level. With this model, we optimize surface coating strategies of nanoparticles to assure good vitreal mobility, while maintaining the normal transfection efficiency. Also, we developed a novel ex vivo retinal model that is bovine‐derived and, in contrast to existing models, keeps the vitreous and the vitreoretinal interface intact. This model is used to identify the potential of nanoparticles to reach retinal cell types after intravitreal injection. As Müller cells are the only cell type connecting the vitreal and retinal side of this interface, we specially focus on the role of Müller cells as our gateway to shuttle intravitreally injected particles across the vitreoretinal interface. As nanoparticles, we focus on polymer‐ or lipid based nanoparticles carrying messenger RNA or plasmid DNA as therapeutic molecules.

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.

An Immunohistochemical Analysis of Hypoxia in Multi-layer Avascular Retina and Vascular Pecten Oculi of the Developing Chicken

The retina lines the internal surface of the back of the eye. In order to function, the retina requires a supply of oxygen. Retinal hypoxia often precedes proliferative diabetic retinopathy in human. In vertebrate eyes, there is a blood supply between retina and choroid, which supports the outer retinal layers. In humans, additional blood vessels enter the retina from the vitreal side and ramify in the plexiform layers. However, in chicks, such plexiform blood vessels are absent. The spatial oxidative state distribution in multi-layer avascular chick retina was studied using the oxygen-sensing reagent EF5. As development of the chick retina proceeds, different oxidation states in different layers were found. The hypoxic staining was almost evenly distributed throughout the tissues for E(mbryonic day) 5 chick retina. For E7 chick retina, the hypoxic stain was concentrated in a subset of ganglion cells. The inner portion of the inner nuclear layer and ganglion cell layer were hypotic for E11 chick retina,. For E14 chick retina, hypoxia was observed at the ganglion cell layer, but additional reaction was seen in the inner portion of the inner nuclear layer and the outer plexiform layer. There was also some reaction in the region of budding photoreceptors. The Ganglion cell layer, and the Inner Nuclear Layer were more hypoxic for E19 chick retina,. In most cases, the regions of hypoxia did not colocalize with the positions that have high mitochondrial density. Pecten oculi is a corrugated flap of tissue that extends along the back of the eye and is embedded in the vitreous body of the avian eye. It also contains a highly efficient glucose transporter. Hypoxia upregulates glucose transport activity. We found that pecten oculi is much less hypoxic than retina tissue nearby. There are more mitochondrial densities in pecten oculi than in retina.

A porous coral-like structure - a new insight into the morphology of the inner limiting membrane of the retina?

By employing scanning (SEM) and transmission electron microscopy (TEM), to identify morphological features of epiretinal samples removed during vitrectomy in patients with lamellar macular hole (LMH) or epiretinal membrane (ERM). We studied 23 eyes of 23 patients divided into two groups. The first group (13 samples, 11 eyes) consisted of patients with LMH; the second (12 samples, 12 eyes) - with ERM. The surgeries yielded a total of 21 epiretinal samples peeled simultaneously with the ILM and 4 epiretinal samples (2 eyes) peeled in two parts, the second part containing the ILM. One half of the samples was studied by SEM without prior dehydratation, the other - by TEM. The study revealed a high degree of ultrastructural similarity between the two groups of ILM samples. Judging from SEM findings, two sides of the membrane were clearly identified. Porous coral-like structures (PCS) were discovered on the side facing the retina. TEM in the area of PCS discovered parallel arrangement of multiple Muller cell (MC) bodies and processes separated by wide layers of the intercellular matrix. The vitreal side of all ILM samples was notable for numerous fibroblast-like cells. Many variously shaped petrified structures were found on both sides of the membrane. During the so called ILM peeling, the surgeon removes a layered structure that includes the basal membrane of MC, cells and fibers attached to its vitreal side, and one more layer comprised by PCS and rather readily torn off from the main massif. The functional significance of this previously unknown structure as well as the effect of its partial removal during surgical manipulations with neurosensory retina in the macular region is yet to be investigated.