The authors have declared that no competing interests exist.
Conceived and designed the experiments: SCB RK XRL JMMH LIL. Performed the experiments: SCB RW. Analyzed the data: SCB RK RW LIL. Contributed reagents/materials/analysis tools: RW. Wrote the paper: SCB RK XRL JMMH LIL.
The vitreoretinal interface is the border of the cortical vitreous and the inner surface of the retina. The adhesion of the cortical vitreous to the ILM, namely vitreoretinal adhesion, involves a series of complex molecular adhesion mechanisms and has been considered as an important pathogenic factor in many vitreoretinal diseases. The presence of type VI collagen at the vitreoretinal interface and its possible interaction with collagens and glycoproteins indicates that type VI collagen may contribute to the vitreoretinal adhesion.
To clarify the ultrastructural location of type VI collagen and its relationship to type II and IV collagens at the vitreoretinal interface.
The ultrastructural localization of type II, IV and VI collagens in the adult human vitreoretinal interface of five donor eyes was evaluated by transmission electron microscopy using immunogold labeling.
In the pre-equatorial region, we observed densely packed vitreous lamellae with a partly intraretinal course containing type II and VI collagens, reticular structures containing type IV and VI collagens and a thin inner limiting membrane (ILM) containing type IV and VI collagens in a linear distribution pattern. From the anterior to the posterior retina, the linear pattern of type IV and VI collagen labeling gradually became more diffusely present throughout the entire thickness of the ILM.
The presence of type VI collagen in vitreous lamellae penetrating the ILM into the superficial retina suggests that type VI collagen may be involved in the organization of vitreous fibers into lamellae and in the adhesion of the vitreous fibers to the retina. The close relation of type VI to type IV collagen in the ILM suggests that type VI collagen is an important collagen type in the ILM. The topographic variations of type IV and VI collagens in the different regions of the ILM suggest a regional heterogeneity of the ILM. The reticular labeling pattern of type IV and VI collagens observed in the anterior vitreous are highly similar to labeling patterns of blood vessel walls. In the anterior vitreous, they may represent remnants of the regressed embryonic hyaloid blood vessel system. Their presence is in support of the theory on interactive remodeling of the developing vitreous as opposed to the main stream theory of displacement and compression of the primary by the secondary vitreous.
The vitreoretinal interface is the border of the cortical vitreous and the inner surface of the retina. It is a complex extracellular matrix (ECM) structure containing cortical vitreous, retinal inner limiting membrane (ILM) and Müller cell endfeet. The major ECM components of the vitreoretinal interface are collagens, glycosaminoglycans (GAGs) and glycoproteins (GPs). Cortical vitreous consists of densely packed heterotypic fibrils containing type II, V/XI and IX collagens. While type II collagen forms the main scaffold of the vitreous body, the other ECM proteins, including type IX collagen, GAGs and GPs, are responsible for stabilizing the collagen network. The chondroitin sulphate chains of type IX collagen and opticin on the surface of the type II collagen fibrils probably maintain the space between the collagen fibrils and prevent their self-aggregation. Hyaluronan is highly hydrated and fills the spaces between the collagen fibrils [
The ILM is essentially the basement membrane of retinal Müller cells and it consists of basement membrane associated ECM proteins of which type IV collagen, laminin, fibronectin and others have been identified [
The adhesion of the cortical vitreous to the ILM, namely vitreoretinal adhesion, involves a series of complex molecular adhesion mechanisms and has been considered as an important pathogenic factor in many vitreoretinal diseases [
Overall and focal vitreoretinal attachment have been studied previously, but only limited information on the specific collagens involved is available. Previous research has indicated that non-collagenous molecules, such as fibronectin, laminin, and opticin are involved in the vitreoretinal adhesion at the posterior pole. These molecules may interact with both type II collagen in the vitreous and type IV collagen in the ILM and help to connect the vitreous to the ILM [
The incomplete knowledge concerning the molecules involved in vitreoretinal adhesion, contributes to making current treatment options for vitreoretinal adhesion related diseases limited and inefficient. Techniques to create a PVD should ensure that a complete PVD is achieved, in order to avoid increased focal vitreoretinal traction, which may exacerbate pre-existing conditions. This can be done surgically, but also pharmacologically. Non-collagenous adhesion molecules have been specifically targeted by injecting enzymes such as chondroitinase, dispase, hyaluronidase and recombinant plasmin into the vitreous [
In previous studies, a number of collagens including type II, V, VI, IX and XI in the vitreous and type IV, VI, VII and XVIII in the ILM have been identified [
In the present study, by using immuno-transmission electron microscopy (TEM), we compared the distribution patterns of type II, IV and VI collagens in the anterior (pre-equatorial), equatorial and posterior regions of the vitreoretinal interface. Hereby, we hope to contribute to the understanding of vitreoretinal adhesion at different anatomic locations.
Five human eyes of 5 donors (35, 56, 66 and 77 years old male, and 78 years old female) without any known ophthalmic disorders were obtained from the Euro Cornea Bank (Beverwijk, the Netherlands. (
The tissue processing and the immuno-TEM procedures were adopted from the protocols of Ponsioen et. al. with some modifications [
The embedded eyes were cut and sections of 3 μm thickness were stained with toluidin blue (TB) for evaluation by LM. The pre-equatorial area, the equator and the posterior pole were selected for morphological and immunohistochemical evaluation by TEM. Sections with a thickness of approximately 200 nm were mounted on formvar-coated nickel grids, subjected to the immuno-TEM procedures described below, and evaluated by a Philips 201 TEM (Amsterdam, the Netherlands) operated at 80 kV.
The 200 nm sections were pretreated with 0.1% trypsin in Tris-HCL (pH 7.8 containing 0.1% CaCl2) for 15 minutes at 37°C. Then, the sections were washed in PBS and incubated in 0.1 M citric acid (pH 3.0) for 30 minutes at 37°C. After washing in PBS, the sections were incubated in PBS with 0.15% glycine, 5% bovine serum albumin (BSA), 2% rabbit serum (for anti-type VI collagen antibody) or 2% goat serum (for anti-type II and IV collagen antibodies) for 30 minutes at room temperature to block the non-specific binding of the primary antibodies. Afterwards, the sections were incubated in the primary antibodies diluted in PBS (1/100) with 1% BSA-c (Aurion, Wageningen, the Netherlands) first for 2 hours at 37°C, and then overnight at room temperature. The primary antibodies included: goat anti-type II collagen (polyclonal, Southern Biotechnology Associates (SBA), Birmingham, USA), goat anti-type IV collagen (polyclonal, SBA) and rabbit anti-type VI collagen (polyclonal, Abcam, Cambridge, UK). The next day, the sections were washed in PBS and incubated in 6 nm gold particles conjugated to secondary antibodies (Rabbit anti Goat IgG or Goat anti-Rabbit IgG; Aurion) diluted in PBS (1/150) for 60 minutes at room temperature. Then, the sections were washed in PBS, incubated in 2% glutaraldehyde in PBS for 2 minutes, and briefly washed in double distilled water. A silver enhancement solution (Aurion R-gent enhancer, Aurion) was then applied for 10 minutes at room temperature. After washing in double distilled water, the sections were counterstained with uranyl acetate in 25 cP Methyl cellulose (MC-UAc, Sigma-Aldrich, St. Louis, USA). The negative controls underwent the entire procedure, except that the primary antibodies were omitted.
To obtain differently sized immunogold labeling to type IV and VI collagens, a three-step immuno-histochemical procedure was used which resulted in the attachment of 15 nm gold particles to type IV collagen, and a two-step procedure with silver enhancement was used to label the type VI collagen with larger particles. Briefly, the 200 nm sections went through the same antigen retrieval processes as aforementioned. The sections were incubated in a mixture of diluted goat anti-type IV collagen and rabbit anti-type VI collagen antibodies (1/100) in PBS with 1% BSA-c (Aurion) at 4°C overnight. On the second day, the sections were washed in PBS and incubated in mouse anti-goat IgG antibody diluted in PBS containing 1% BSA-c (Aurion) for 30 minutes. After washing in PBS, the sections were incubated in 6 nm gold particles conjugated to goat anti-rabbit IgG antibody (Aurion) diluted in PBS containing 1% BSA-c (Aurion) at room temperature for 45 minutes. Afterwards, the sections were washed in double distilled water and incubated in silver enhancement kit (Aurion) for 10 minutes at room temperature. This procedure resulted in the immunogold labeling of type VI collagen with silver enhancement, thus creating relatively large electron dense particles. Subsequently, the sections were washed in double distilled water and incubated in 15 nm gold particles conjugated to goat anti-mouse IgG antibody diluted in PBS containing 1% BSA-c (Aurion) at room temperature for 45 minutes. Thus, type IV collagen was labeled by the 15 nm gold particles. After washing in double distilled water, the sections were counterstained with uranyl acetate in 25 cP MC-UAc at 4°C for 15 minutes.
The ILM contains densely packed filaments forming an interwoven network. The filamentous network configuration of the ILM was most prominent at the pre-equatorial and equatorial areas. At the posterior region, the ILM is thickened and indented at its retinal surface. The vitreous surface of the ILM does not have an entirely smooth aspect, but it has numerous branching filaments extending themselves towards the vitreous body.
Pre-equatorial area: The majority of vitreous fibers are condensed into lamellae. Vitreous fibers attach themselves to the ILM but they also penetrate the ILM and attach themselves to the superficial retina. At the pre-equatorial area, the ILM is thin and it has an electron density that is comparable to that of the retina, which makes it difficult to clearly delineate the two tissues (Figs
Equatorial area: At the equatorial ILM, vitreous fibers were observed in the 35 and 66 years old donors (Figs
Posterior pole: The ILM was thicker in this area compared to the pre-equatorial and equatorial regions. Furthermore, retinal indentations of the ILM became more prominent both in number and extent (Figs
A. Eye of a 56 year-old donor. Immunogold labeling directed at type II collagen was located on individual vitreous fibers in the basal vitreous and superficial retina. Type II collagen positive fibers in the basal vitreous were often seen to condense and form lamellar structures (arrow). Vitreous fibers penetrated the ILM, which is indicative of attachment of these fibers to the superficial retina (arrow head). B. Eye of a 35 year-old donor. Vitreous fibers containing immunogold labeling to type II collagen at the equator. Note that gold labelled fibers penetrate the ILM and attach themselves to the superficial retina. C. Eye of a 66 year-old donor. At the posterior vitreoretinal interface, immunogold labeling directed at type II collagen was located on the cortical vitreous fibers (arrows) and the vitreal side of the inner limiting membrane (arrow heads). Bar = 2μm. V = vitreous; R = retina.
A. Pre-equatorial ILM of the eye of the 56 year-old donor. Note the linear distribution pattern of the labeling. B. Equatorial ILM of the eye of the 77 year-old donor. Note the more diffuse staining pattern. C. Posterior vitreoretinal interface of the 56 year-old donor eye. Type IV collagen appears to be distributed throughout the entire thickness of the ILM. A and B, bar = 1 μm; C, bar = 5 μm. V = vitreous; R = retina.
A. In the eye of the 56 year-old donor, type VI collagen labeling was found on the vitreous lamellae that run perpendicular to the pre-equatorial retina (black arrow heads), on fine cortical vitreous fibers (asterisks) and in the superficial retina (white arrows). B. In the eye of the 77 year-old donor, type VI collagen was observed at the equatorial ILM in a thicker than just linear band (black arrows). C. In the eye of the 56 year-old donor, type VI collagen was distributed throughout the entire thickness of the posterior ILM (black arrows). V = vitreous; R = retina. Bar = 1 μm.
Type II collagen staining of vitreous fibrils at the pre-equatorial (A), equatorial (B) and posterior (C) vitreoretinal interface. Type IV collagen staining of the ILM at the pre-equatorial (D), equatorial (E) and posterior (F) vitreoretinal interface. Note the lineair labeling pattern at the pre-equatorial location, and the more diffuse distribution of the labeling at the equator and posterior sites. Type VI collagen staining of the ILM and cortical vitreous at the pre-equatorial (G), equatorial (H) and posterior (I) vitreoretinal interface. The ILM thickens towards the posterior retina. In this younger donor, the ILM at the posterior pole is thinner than that in the older donor (compare with
Type II collagen staining of vitreous fibrils at the pre-equatorial (A), equatorial (B) and posterior (C) vitreoretinal interface. Type IV collagen staining of the ILM at the pre-equatorial (D), equatorial (E) and posterior (F) vitreoretinal interface. Type VI collagen staining in the pre-equatorial (G), equatorial (H) and posterior (I) vitreoretinal interface. Note the lineair labeling pattern at the pre-equatorial location, and the more diffuse distribution of the labeling at the equator and posterior sites. D and G: note reticular pattern (arrows). Also, the ILM thickens towards the posterior retina, in this older donor, the ILM at the posterior pole is thicker than that in the younger donor (compare with
A. Immunogold labeling of type IV collagen displayed a reticular pattern at the pre-equatorial vitreoretinal interface (square box). Insert: Detail of marked area in A: Type IV collagen positive structures in the vitreous extending themselves to the adjacent retina (black arrows). B: Linear distribution of type IV collagen indicating the location of the ILM (arrows) and type IV collagen positive structures in the vitreous attaching themselves to the ILM and superficial retina (black arrow heads). C. Distribution of type IV collagen in the basement membrane of a choroidal blood vessel displaying a similar reticular pattern as that found in the pre-equatorial intravitreal reticular structures (black arrow heads). A, bar = 20 μm, insert, bar = 2 μm; B, bar = 2 μm; C, bar = 1 μm. V = vitreous; R = retina.
A. Overview of the pre-equatorial vitreoretinal interface stained for type VI collagen. Insert: Detail ofmarked area in A: Type VI collagen forms reticular structures in the vitreous (arrows) that extend themselves towards the superficial retina (arrow heads). B: Type VI collagen forms a reticular pattern in the superficial retina at the pre-equatorial area (arrow heads). C. Type VI collagen in the basement membrane of a retinal blood vessel displayed a pattern similar to that found in the pre-equatorial reticular intravitreal structure (arrow heads). A, bar = 10 μm, insert, bar = 1 μm; B and C, bar = 1 μm. V = vitreous; R = retina.
The overall localization of type II, IV and VI collagens in the vitreoretinal interface was summarized in
Pre-equatorial area | Equator | Posterior pole | |||||||
---|---|---|---|---|---|---|---|---|---|
Vitreous | ILM | Inner retina | Vitreous | ILM | Inner retina | Vitreous | ILM | Inner retina | |
Type II collagen | (+) | (-) | (+) | (+) | (-) | (+) | (+) | (-) | (+) |
Type IV collagen | (+) |
(+) | (-) | (-) | (+) | (-) | (-) | (+) | (-) |
Type VI collagen | (+) | (+) | (+) | (+) | (+) | (-) | (+) | (+) | (-) |
ILM: inner limiting membrane;
*type IV collagen is attached to reticular structures in the anterior vitreous.
Immunogold labeling directed at type II collagen was identified on the vitreous fibers. At the pre-equatorial region of all donor eyes, the vitreous was attached to the retina and vitreous lamellae and individual vitreous fibers were found to penetrate the ILM and to attach themselves to the underlying retina. Gold labeling was observed in a substantial part of the superficial retina (Figs
At the pre-equatorial and equatorial areas, immunogold labeling directed at type IV collagen displayed a linear configuration (Figs
At the pre-equatorial area, immunogold labeling directed at type VI collagen was diffusely distributed throughout the cortical vitreous and was frequently identified to be concentrated in a linear pattern alongside the densely packed vitreous lamellae which attached themselves to the basal retina (
Reticular structures containing both type IV and VI collagens were identified at the pre-equatorial region of the vitreoretinal interface. These reticular structures were located intra-vitreally, were attached to the ILM and extended themselves into the superficial retina. The labeling pattern was highly similar to that found in basement membranes of retinal and choroidal blood vessels (Figs
The double labeling of type IV and VI collagens displayed a similar distribution pattern: a linear labeling pattern at the pre-equatorial ILM, and a gradually more diffuse distribution of labeling in the direction of the posterior ILM. Additionally, an intense staining in the basement membranes of retinal and choroidal blood vessels was observed. Furthermore, the 6 nm gold label with silver enhancement directed to type VI collagen could be detected on the vitreous fibrils (
The large labels are gold particles with silver enhancement representing type VI collagen (arrows). The small labels are 15 nm gold particles representing type IV collagen (arrow heads). A. In the eye of the 56 year-old donor, the equatorial area exhibits a linear distribution pattern of both type IV and VI collagens. Note that type VI collagen can also be seen on the vitreous fibrils. B. In the eye of the 35 year-old donor, towards the posterior pole, both type IV and VI collagens become diffusely distributed over the entire thickness of the ILM. A, B bar = 1 μm. V = vitreous; R = retina.
The current immuno-TEM study expands on previous studies by providing detailed information on the site-specific ultrastructural distribution of type II, IV and VI collagens at the vitreoretinal interface. These types of collagen are not restricted to either the vitreous or the retina, but encompass a wider area at the vitreoretinal interface. Thus, they are likely involved in vitreoretinal attachment. Type IV and VI collagens have similar distribution patterns at the ILM, whereas type VI collagens can also be found diffusely in the vitreous cortex and located to intravitreal lamellae. Type IV and VI collagens stain reticular-like structures at the vitreous base. These labeling patterns are highly similar to those found in retinal and choroidal blood vessel walls, which indicates a vascular origin of these structures.
At the pre-equatorial vitreoretinal interface, we observed densely packed vitreous lamellae with an intraretinal course containing type II and VI collagens, reticular-like structures containing type IV and VI collagens that span a wide vitreoretinal area and a thin ILM containing type IV and VI collagens in a linear distribution pattern.
In previous studies, type VI collagen was mainly found in the connective tissue closely associated with basement membranes and it was proposed to function as an anchoring fiber [
Type IV and VI collagens are known components of the basement membrane of blood vessels [
The residual vascular remnants found at the pre-equatorial vitreoretinal interface support the theory that the secondary vitreous is formed by a process of interactive remodeling of the primary vitreous [
This hypothesis on the development of the secondary vitreous, is in conflict with the main stream theory. The main stream theory proposed that the avascular–secondary–vitreous body starts to form between the primary vitreous and retina, and surrounds and compresses the primary vitreous. While the secondary vitreous gradually increases its volume, the primary vitreous ceases to grow and regresses. Based on this theory, the vascular remnants would be located exclusively in the center of the vitreous. This theory is supported by the observation of large vessel remnants in the center of the vitreous sometimes found by slit lamp microscopy and optical coherence tomography [
Previous studies already found a regional variability in morphological aspects of the ILM. These observations were confirmed in the present study and consisted of a thin ILM anteriorly without indentations, and thickening of the ILM towards the posterior pole with the development of indentations at its retinal side. Also, anteriorly, the electron density of the ILM was comparable to that of the subjacent retina, whereas the ILM towards the posterior part of the eye has a higher electron density than the subjacent retina. The present study expands on existing knowledge by clarifying the site-specific arrangement of type IV and VI collagens at the retinal ILM. The linear labeling pattern of type IV and VI collagens at the pre-equatorial ILM changes into a more diffuse labeling of the entire thickness of the ILM towards the posterior pole. This suggests a regional heterogeneity of this basement membrane. Such heterogeneity would be consistent with previous publications on regional differences in collagenous composition of basement membranes in organs such as kidney and lung [
Type IV collagen is the predominant ECM protein in human ILM which accounts for about 57% of the total proteins [
Previous studies showed that type VI collagen forms a fine beaded filament network in the vicinity of basement membranes, whereas the expression of type VI collagen in the basement membrane was only reported in the renal glomerular basement membrane and intestinal epithelial basement membrane [
The presence of type IV and VI collagens throughout the entire thickness of the posterior ILM, suggests that these collagens are synthesized during the aging process. In our series as well as in previous studies, the ILM has been shown to gradually thicken towards the posterior pole with increased numbers of ILM indentations at its retinal side. These extensions of the ILM, as suggested by previous authors, are considered to be age-related depositions of ECM onto the retinal aspect of the ILM [
It has been suggested that the majority of the ILM proteins such as type IV collagen and laminin, are produced by lens and ciliary body epithelium and are then transported through the vitreous during the embryonic period [
The immuno-TEM technique allows the target protein to be localized with high precision. This technique has found wide application in studying the ultrastructure of the basement membrane and its adjacent ECM. However, the procedure has certain limitations which should be taken into account. One of the important issues is the specificity of the antibodies. In this study, we used commercially available antibodies and applied a double labeling technique to type IV and VI collagens to reduce the chance of false interpretations due to antibody cross-reaction. We observed type IV and VI collagen labeling at different sites within the ILM and the vascular basement membrane. In addition, type VI collagen labeling was observed on vitreous fibers and lamellae, whereas type IV collagen labeling on these structures was absent. Combined with the results of the negative controls that ran parallel with each immuno-TEM procedure, it is likely that the immunogold labeling directed at type II, IV and VI collagens is specific.
In conclusion, our findings concerning the ultrastructural distribution of type II, IV and VI collagens indicate that the distribution of collagens in the ILM is heterogeneous. Type IV and VI collagens may form a single layer network in the pre-equatorial ILM and encompass the entire thickness of the ILM towards the posterior pole. Immunogold labeling for type IV collagen was exclusively found in the basement membrane and on the reticular structures in the anterior vitreous, whereas type VI collagen was additionally observed on vitreous lamellae and fibrils closely associated with the ILM. These findings indicate that type VI collagen could function as an anchoring fibril to organize vitreous fibers into lamellae and to attach them to the ILM and the underlying retina. The reticular labeling patterns of type IV and VI collagens support the hypothesis of interactive remodeling of the vitreous during its embryonic development.
At the retina of a 77 year-old donor without any known ophthalmic disorders, a fibrocellular proliferation (epiretinal membrane, ERM) containing type IV (
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The ERM containing type VI collagen positive fibers showed focal attachments to the ILM (arrow heads). ERM = epiretinal membrane; ILM = inner limiting membrane. Bar = 1μm.
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The fibrocellular membrane lies on the vitreal side of the inner limiting membrane (ILM), which is positive to the antibody against type IV collagen. The type IV collagen staining displayed a linear pattern in the ERM (arrows) and a diffuse pattern in the ILM. ILM = inner limiting membrane; EC = epiretinal cell; V = vitreous; R = retina. Bar = 2μm.
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