Vitreous Collagen Research Articles

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.

Age-related cataract and macular degeneration: Oxygen receptor dysfunction diseases

Age-related cataract and age-related macular degeneration (AMD) is the leading cause of vision impairment and blindness in developing and developed countries, respectively. Oxidative stress and oxidation products have been verified to play important roles in these two aging diseases. Recent research has demonstrated that there are significant oxygen gradients in the eye. Therefore, we propose a new hypothesis that these two diseases could be summarized as oxygen receptor dysfunction diseases of which the main points are as follows. Oxygen in the retinal and choroidal vasculature is transferred into the vitreous cavity by a special switching valve or oxygen receptor that might exist in the internal limiting membrane, vascular endothelium or posterior vitreous surface. It is then transported from the posterior segment to the anterior segment by vitreous collagen fibrilla networks, which work similar to a gas pipeline. Posterior vitreous detachment is the starting point of these two diseases by inducing formation of the local hyperoxia region, which results in the occurrence of age-related cataract and macular degeneration. Thus, an innovative anti-oxidative therapy should be added to the traditional treatment of age related macular degeneration. Some associated experimental and clinical approaches are suggested in our paper to test this hypothesis.

The role of extracellular matrix in retinal vascular development and preretinal neovascularization

Extracellular matrix (ECM) plays a central role in angiogenesis. ECM degrading enzymes breakdown the pre-existing vascular basement membrane at an early stage of angiogenesis and subsequently degrade stromal ECM as the new vessels invade into tissues. Conversely certain ECM components including collagen, fibronectin or fibrin are required for endothelial cell migration and tube morphogenesis. As the new vessels form they lay down a basement membrane that surrounds the endothelial tubes and is essential for their stability. In the rodent eye the transient expression of fibronectin and matricellular proteins plays a key role in retinal vascular development. In pathological retinal angiogenesis, such as in proliferative diabetic retinopathy, preretinal neovascularization occurs where new blood vessels invade the cortical vitreous gel and these blood vessels require vitreous collagen for their growth. The vitreous is normally anti-angiogenic and contains endogenous ECM inhibitors of angiogenesis including opticin and thombospondins, and ECM fragments such as endostatin. In preretinal neovascularization, the combined anti-angiogenic effects of these molecules are overcome by an excess of growth factors such as vascular endothelial growth factor-A, and new vessels grow into the vitreous with potentially blinding sequelae.

Clinicopathological correlations at the vitreoretinal interface

Clinicopathological studies of the vitreoretinal interface (VRI) improve our understanding of the pathogenesis of vitreal maculopathy, facilitate differential diagnoses and help to develop new treatment strategies. The aim of the study was to provide a comprehensive overview on clinicopathological correlations of the VRI. A semi-structured literature search was performed in the Medline and Embase databases for relevant original studies on clinicopathological correlations of vitreal maculopathy, in addition to the latest books and review articles. Age-related vitreous changes with persistent vitreomacular adhesions on the retinal surface promote cellular migration and proliferation onto the vitreal side of the internal limiting membrane (ILM), thereby cementing the vitreomacular adhesions and strengthening the traction forces on retinal layers. Cellular or fibrocellular proliferation at the vitreomacular interface can be seen in all vitreal maculopathies. Furthermore, vitreoschisis in the context of anomalous posterior vitreous detachment causes the presence of vitreous cortex collagen fibrils on the vitreal side of the ILM which is associated with epiretinal membrane formation. Glial cells, hyalocytes and myofibroblasts represent the major cell types in the epiretinal cell proliferation. Glial cells and hyalocytes are capable of transdifferentiation into myofibroblasts which possess strong contractive properties and are well known for the production of extracellular matrix components. Removing vitreomacular adhesions and vitreous cortex collagen fibrils from the retinal surface is most important for successful treatment. In cases with epiretinal cell proliferation, however, removal of the ILM during macular surgery is mandatory to avoid reproliferation and recurrence. Improving the detection of epiretinal cell proliferation and cell distribution in patient eyes by optical coherence tomography or by introduction of new technologies should be addressed in the future.

Correlative Microscopy of Lamellar Hole-Associated Epiretinal Proliferation.

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

Hyalocytes in idiopathic epiretinal membranes: a correlative light and electron microscopic study.

To describe characteristics of epiretinal cells at the vitreoretinal interface by correlative light and electron microscopy (CLEM). Epiretinal membrane (ERM) specimens and internal limiting membrane (ILM) specimens were harvested by sequential peeling during vitrectomy from 27 eyes with idiopathic epiretinal gliosis, and processed for CLEM. Intraoperatively, the presence of posterior vitreous detachment (PVD) was documented. We used anti-vimentin, anti-α-smooth muscle actin (α-SMA), and anti-CD45 as primary antibodies. A fluorescein-tagged immunonanogold cluster was used as secondary antibody and visualized under the fluorescence and transmission electron microscope. We demonstrated CD45-positive cells specifically labelled at their plasma membranes with ultrastructural features known for hyalocytes, such as oval nucleus with marginal chromatin, vacuoles, dense granules, and thin cytoplasmic protrusions. CD45-positive cells were mostly located on a thick layer of native vitreous collagen. They were covered by newly formed collagen strands with multilayered proliferation of myofibroblasts. We also demonstrated immunoreactivity for vimentin and alpha-SMA. Cell fragments with positive labelling for α-SMA and vimentin were not only found on the vitreal side of the ILM, but also on the retinal side. By CLEM, the majority of CD45-positive cells in epiretinal cell proliferation were characterized as hyalocytes. In the context of anomalous PVD and vitreoschisis, ultrastructural features and topographic localization of hyalocytes suggest that these cells play a significant role in ERM formation. CLEM enables a more accurate characterization of epiretinal cell proliferation, and therefore, contributes to a better understanding of the pathogenesis of diseases at the vitreoretinal interface.

Pathophysiology of vitreoretinal interface disorders

AbstractPathologic changes at the vitreoretinal interface and vitreomacular tractions lead to phenotypic lesions such as epiretinal membranes (ERMs), idiopathic macular holes, vitreomacular traction syndrome (VMTS) and myopic foveoschisis. In the presence of clinically evident posterior vitreous (PVD), remnants of vitreous collagen fibers and/or cellular proliferations of fibrous astrocytes or glial cells, migrate outward from the retina. Migration occuring on the inner vitreo‐retinal interface through the internal limiting membrane(ILM), in the presence of growth factors such as laminin or fibronectin, may contribute to the fibrocellular proliferation at the retinal surface. Most studies regarding idiopathic or diabetic epiretinal membranes or late stages of macular holes, described the presence of fibrocytes or myofibroblasts. More recent studies confirmed those observations by using antibodies against alpha smooth muscle actine (a‐SMA). Based on the mechanistic association between a‐SMA expression and a tractional force generation, there is little doubt that these cells represented the source of traction during the evolution of those pathologies.

Pharmakologische Vitreolyse

Posterior vitreous detachment (PVD) is a physiological ageing process. In many cases PVD is incomplete and pathological adhesions of vitreous collagen may be associated with tractional forces in the periphery where they can cause retinal breaks and lead to detachment of the neurosensory retina. In the macular area such tractional forces at the vitreoretinal interface can contribute to the formation of specific entities such as vitreomacular traction syndrome and macular holes which are associated with an impairment of visual acuity and disturbing metamorphopsia. Currently, pars plana vitrectomy with induction of PVD, peeling of epiretinal membranes and the ILM represents an effective and safe treatment option for these conditions. Pharmacological vitreolysis is a new and alternative, non-surgical approach to release tractional forces at the vitreoretinal interface by injecting an enzyme with proteolytic activity against fibronectin and laminin into the vitreous cavity. Certain forms of vitreomacular traction and smaller macular holes can be successfully treated in this way without surgical manipulation of the retinal surface. The role of the concept of pharmacological vitreolysis as a treatment option even for exudative macular diseases or as an adjunct to assist vitreoretinal surgical procedures is currently under investigation.

Normal and abnormal vitreoretinal interface conditions

AbstractPathologic changes at the vitreoretinal interface and vitreomacular traction, lead to the phenotypic lesions as epiretinal membranes (ERMs), idiopathic macular holes, vitreomacular traction syndrome (VMTS) and myopic foveoschisis. Remnants of vitreous collagen fibers in the presence of clinically evident posterior vitreous detachment (PVD) and/or cellular proliferations of fibrous astrocytes or glial cells growing outward from the retina to the inner retinal surface through the internal limiting membrane (ILM) , in the presence of growth factors as laminin, fibronectin, may contribute to fibrocellular proliferation at the retinal surface. Most studies of idiopathic of diabetic or macular holes related epiretinal membranes, described the presence of fibrocytes or myofibroblasts and more recent studies confirmed these observations using antibodies against alpha smooth muscle actine (a‐SMA). Based on the mechanistic association between a‐SMA expression and tractional force generation, there is little doubt that these cells represented the source of traction during the evolution of those pathologies.

Inhibition of PVR with a Tyrosine Kinase Inhibitor, Dasatinib, in the Swine

We tested the efficacy of dasatinib, a Food and Drug Administration (FDA)-approved tyrosine kinase inhibitor, to prevent proliferative vitreoretinopathy (PVR). The effect of dasatinib on RPE sheet growth was determined by measuring enlargement of cultured RPE sheets in the presence or absence of dasatinib. Epithelial-mesenchymal transition (EMT) of RPE cells was assessed by expression of S100A4. A scratch wound assay, cell number count, and type I collagen contraction assay were used to examine the effect of dasatinib on migration, proliferation, and extracellular matrix (ECM) contraction, respectively. Our swine model of experimental PVR with green fluorescent protein-positive (GFP+) RPE cells was used to assess the efficacy of dasatinib in preventing traction retinal detachment (TRD) caused by PVR. Full-field electroretinography and histologic examination were used to determine the retinal toxicity of dasatinib. Dasatinib prevented RPE sheet growth, cell migration, proliferation, EMT, and ECM contraction in a concentration-dependent manner. 0.1 μM dasatinib inhibited nearly 80% of vitreous fluid-stimulated collagen gel contraction. Dasatinib also prevented TRD caused by PVR in vivo. Only 1/11 eyes had a TRD in the presence of dasatinib, while all 11 controls eyes had a TRD. Dasatinib did not cause any detectable toxicity of the retina. Dasatinib significantly inhibited PVR-related RPE changes in vitro and prevented TRD in an experimental PVR model in the swine without any detectable toxicity. Our data suggested that dasatinib may be effective in the prevention of PVR.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Lamellar Macular Hole: A Clinicopathologic Correlation of Surgically Excised Epiretinal Membranes

To correlate clinical and optical coherence tomographic (OCT) features with morphologic and immunohistochemical findings of epiretinal membranes (ERMs) in lamellar macular holes (LMHs). Nineteen specimens were removed from 19 eyes during vitrectomy for lamellar macular hole with ERM and internal limiting membrane peeling, and were processed for transmission electron microscopy and immunohistochemistry by cross-sectional and flat-mount preparation techniques. By using OCT criteria and intraoperative observations, ERM specimens were divided into two groups: 13 "dense" and 6 "tractional" membranes. Patients' records were reviewed. "Dense" ERMs were seen with abundant clusters of fibrous long-spacing collagen embedded in compactly folded native vitreous collagen strands. Posterior hyaloids were attached to the retina in the majority of cases. Both groups of ERMs showed positive immunoreactivity for glial fibrillic acidic protein and hyalocyte markers. Anti-α-smooth muscle actin labeling was most positive in "tractional" ERMs. Surgery resulted in significant improvement (74%) of visual acuity, with a mean gain of 2 Snellen lines in both groups. All other patients (26%) preserved vision. Three patients (16%) developed a full-thickness macular defect requiring additional surgery. Morphologic components differ in epiretinal cell proliferations of LMHs. In association with degradation of vitreous collagen, glial cells and hyalocytes seem to play an important role in LMH development. Since functional benefit after surgery was limited and progression to a full-thickness macular defect was a notable complication, we recommend caution in proceeding with surgical intervention. Further investigations are needed to elucidate whether "dense" and "tractional" LMHs represent different disease entities or different stages of one disorder.

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...

Macular appearance after diabetic vitrectomy for fibrovascular proliferation: an optical coherence tomography study

To investigate morphological variations in the macular area with optical coherence tomography (OCT) after vitrectomy for diabetic fibrovascular proliferation. We reviewed 108 cases using OCT 7-15 months after vitrectomy. Of these, 32 received OCT within 3 months postoperatively. Morphological variations were categorized and correlated with visual outcome. Only 24 cases (21.4%) had no obvious abnormalities. The most frequent findings were epiretinal membrane (52.8%), macular thickening (37.0%) and macular cysts (28.7%). Multivariate regression showed that diffuse macular thickening, loss of foveal depression and diffuse retinal thinning were significantly associated with poor visual acuity. Sequential OCT (< 3 and >or= 7 months) revealed that epiretinal membrane and oedema outside of fovea changed significantly between two examinations. OCT may identify diverse morphological changes in the macular area after diabetic vitrectomy for fibrovascular proliferation. Macular appearance may change over time, and certain types of morphological changes may be associated with poor visual function.

Insights into the molecular basis of rhegmatogenous retinal detachment

Abstract Purpose Factors that determine the likelihood of developing posterior vitreous detachment and subsequent rhegmatogenous retinal detachment (RRD) include (i) the degree of vitreous liquefaction (ii) the strength of post‐basal vitreoretinal adhesion and (iii) the topology of the posterior border of the vitreous base. The purpose of these studies was to investigate each of these using a combination of ultrastructural and molecular techniques. Methods Ultrastructural studies of the human vitreous and vitreoretinal interface were performed in combination with various antibodies and cationic dyes. Biochemical studies were performed on extracted vitreous components. Results The resultant data suggest that: (i) vitreous liquefaction is caused by the aggregation of vitreous collagen fibrils and this is due to a loss of type IX collagen proteoglycan from the fibril surfaces; (ii) interactions between heparan sulphate proteoglycans in the inner limiting lamina and components on the surface of cortical vitreous collagen fibrils contribute to postbasal vitreoretinal adhesion; (iii) the posterior border of the vitreous base migrates posteriorly with aging due to the synthesis of new vitreous collagen by the peripheral retina. Conclusion The molecular basis of RRD is starting to be unravelled. Furthering our understanding of the underlying molecular processes may lead to the development of novel therapeutic strategies to treat RRD and other vitreoretinal disorders.

PATHOLOGY OF THE MACULAR HOLE RIM IN FLAT-MOUNTED INTERNAL LIMITING MEMBRANE SPECIMENS

The purpose of this study was to image the pathology of vitreoretinal adhesion and cellular proliferation at the macular hole rim for a better understanding of macular hole formation. Internal limiting membrane (ILM) peeling, ILM flat-mount preparation, phase contrast and interference microscopy, transmission electron microscopy, and immunocytochemistry of the macular hole rim were performed in 10 eyes with idiopathic macular holes. Phase contrast and interference microscopy showed cellular proliferation on the vitreal side of the ILM in all specimens. There were single cells in three eyes, cell clusters in six eyes, and continuous proliferation in one eye. Surrounding the macular hole, only a few cells were present. Cellular proliferation started at a distance of 80 mum to 940 mum from the macular hole edge. Transmission electron microscopy showed vitreous adhesion at the macular hole rim. In ultrastructural terms, there was a continuous insertion of vitreous collagen fibers into the ILM. Immunocytochemistry was positive for collagen type II, laminin, and fibronectin in the area of vitreous attachment. Cells were stained with markers for glial cells, retinal pigment epithelial cells, and hyalocytes. There is ultrastructural evidence that macular hole formation is caused by an insertion of the cortical vitreous into the foveal ILM. Vitreous collagen fibers may exert vitreofoveal traction, resulting in a foveal tear. Cellular proliferation is not continuously distributed surrounding the macular hole but separated from the macular hole edge and seems to originate from cell clusters forming centers of proliferation distant from the macular hole rim.

Pentosidine accumulates in the aging vitreous body: A gender effect

The human vitreous body undergoes structural changes with aging. This can be followed by a posterior vitreous detachment, which can result in ocular pathology. As in many collagenous tissues, age-related changes in the vitreous could be caused by the formation of advanced glycation end products (AGEs). The goal of this study was to find out whether the AGE pentosidine accumulates in the human vitreous with aging. With this data we were able to estimate the half-life of vitreous collagen. Furthermore, we analyzed whether there was a gender difference in pentosidine accumulation, as this was seen in other tissues as well. Using high performance liquid chromatography, pentosidine contents were determined in whole vitreous bodies and in separate parts of vitreous bodies, which were all obtained from human donor eyes. Our results show that pentosidine accumulates in the human vitreous. From the rate of accumulation we could roughly estimate that vitreous collagen has as a similar or shorter half-life compared to skin collagen. This supports the concept of collagen turnover in the vitreous. In general, the female vitreous experiences a faster pentosidine accumulation than the male vitreous, and most of the pentosidine accumulation in the former occurs after 50 years of age.

Analysis of the vitreous membrane in a case of type 1 Stickler syndrome

Stickler syndrome causes ocular abnormalities, including retinal detachment and vitreoretinal degeneration, and systemic anomalies such as arthritis and deafness. Although retinal detachment is characteristic of this syndrome, the pathogenesis is unknown. A 10-year-old boy reported decreased vision and presented 5 days after visual loss. Ophthalmoscopy showed a retinal detachment with a giant tear in the right eye, and a nonpigmented epithelial detachment with pars plicata breaks in the left eye. Bilateral findings included an empty vitreous and a vitreous membrane at the equator. The systemic abnormalities included short stature and joint hypermobility. The diagnosis was type 1 Stickler syndrome, and the eyes were treated surgically. Immunohistochemistry showed that the vitreous membrane resected intraoperatively was comprised primarily of Müller cells. Electron microscopy showed dense collagen fibers around the cells in the membrane that were identical to the vitreous collagen inserted into the basement membrane of the cells, which was similar to the ultrastructure of the vitreous base. Müller cells might be primary components of the vitreous membrane in type 1 Stickler syndrome. The vitreoretinal interface, which resembled the ectopic vitreous base, in the vitreous membrane may be related to the pathogenesis of the retinal detachment.

Adult vitreous structure and postnatal changes

This review will focus on the molecular organisation of the adult vitreous and how it undergoes ageing changes throughout life that result in vitreous liquefaction and a predisposition towards posterior vitreous detachment and retinal break formation. At birth, the vitreous humour is in a gel state due to the presence of a network of fine collagen fibrils. With ageing, these collagen fibrils progressively aggregate due to a loss of type IX collagen from their surfaces. The aggregation of collagen fibrils may cause vitreous liquefaction which, when combined with an age-related weakening of postbasal vitreoretinal adhesion, predisposes to posterior vitreous detachment. Throughout postnatal life, the posterior border of the vitreous base migrates posteriorly from the ora serrata into the peripheral retina. This is due to new collagen synthesis by the peripheral retina. This new collagen intertwines with pre-existing cortical vitreous collagen to create new adhesions and thereby extends the vitreous base posteriorly. If irregularities in the posterior border of the vitreous base arise from this process, there is a predisposition towards retinal break formation during posterior vitreous detachment and subsequent rhegmatogenous retinal detachment.

Ultrastructure of the Vitreomacular Interface in Full-Thickness Idiopathic Macular Holes: A Consecutive Analysis of 100 Cases

To evaluate the ultrastructure of the vitreoretinal interface in stage III and stage IV idiopathic macular holes. A consecutive observational case series, laboratory investigation. Pars plana vitrectomy with en-bloc removal of the internal limiting membrane (ILM) and epimacular tissue was performed by one surgeon in 80 eyes with stage III macular holes and in 20 eyes with stage IV macular holes. In total, 218 specimens were processed for light and transmission electron microscopy. Fibrocellular proliferation at the vitreal side of the ILM was found in 57 cases. Native vitreous collagen (NVC) was attached to the ILM in 36 eyes. The presence of NVC was considerably more frequent in eyes with stage IV (70%) than in eyes with stage III macular holes (26%). Mono- and multilayered cellular membranes were seen more frequently in stage IV macular holes. NVC, if present, was always associated with fibrocellular proliferation. In 39 eyes with stage III and in four eyes with stage IV macular holes, the ILM was devoid of any cells and collagen. Fibrocellular proliferation appears to be a secondary event instead of a primary feature in macular hole development. The severity of fibrocellular proliferation is associated with the presence of NVC. Incomplete vitreoretinal separation may contribute to the development of epimacular membranes in eyes with idiopathic macular holes. Remnants of the vitreous cortex remain more often attached to the ILM in eyes with spontaneous posterior vitreous detachment (PVD) than in eyes with surgical PVD induction.