Although vitreous is the largest structure within the eye, comprising 80% of its volume, our knowledge of vitreous structure and function is perhaps the least of all ocular tissues. Historically, investigations of vitreous structure have been hampered by two fundamental difficulties: first, any attempts to define vitreous morphology are attempts to visualize a tissue that is invisible by design (Fig. 1).¹ Considerable barriers must be overcome to adequately study the structure of an invisible tissue. Second, the various techniques that were used previously to define vitreous structure were fraught with artifacts that biased the results of these investigations. Thus, as noted by Baurmann² and Redslob,³ histologic studies performed during the nineteenth and early twentieth centuries were flawed by the use of tissue fixatives that caused the precipitation of what we recognize today as the glycosaminoglycan (GAG) hyaluronan (HA; formerly called hyaluronic acid).

The development of slit-lamp biomicroscopy by Gullstrand⁴ in 1912 generated a great deal of excitement because it was anticipated that this technique would enable investigations of vitreous structure without the introduction of artifacts. Yet as noted by Redslob,³ a varied set of descriptions resulted, mostly because of the first of the previously mentioned inherent difficulties (i.e., vitreous is largely invisible). This problem even persists in more recent investigations. Eisner⁵ has described membranelles; Worst,⁶ cisterns; Sebag and Balazs,^7,8 fibers; and Kishi and Shimizu,⁹ pockets in the vitreous body. It is likely that the membranelles described by Eisner are composed of the collagen fibers described by Sebag and Balazs. The observation of so-called pockets by Kishi and Shimizu has been explained as the result of age-related liquefaction of vitreous gel, with little relevance to the normal macromolecular structure.^10,11

A clear concept of vitreous structure can only derive from an accurate understanding of its macromolecular organization. The following describes the molecular constituents of vitreous and the macromolecular organization that results in the exquisite anatomic structure of vitreous. Aging and disease-related changes are described as they pertain to the pathophysiology of the major vitreoretinal disorders^12,13 as well as the rationales of surgical intervention¹⁴ and preventative^15,16 modalities.

MOLECULAR CONSTITUENTS

Collagens

Collagen is an important structural protein in vitreous, as in connective tissues everywhere. Gloor¹⁷ pointed out that the collagen content is highest where the vitreous is a gel. Gross¹⁸ was the first to claim that vitreous collagen fibrils are morphologically distinct from collagen in other connective tissues. Yet, Swann and co-workers¹⁹ demonstrated that the amino acid composition of the insoluble residue of vitreous is similar to that of cartilage collagen and later identified that it is most similar to cartilage collagen composed of α1 type II chains.²⁰ Comparisons of the arthritogenic and immunologic properties of collagens from bovine articular cartilage (type II) and vitreous showed that the two were indistinguishable by these assays.²¹ However, subsequent studies²² demonstrated that while vitreous collagen contains an α1 type II chain similar to cartilage collagen, there is a lower alanine content. Furthermore, these studies found that vitreous collagen has additional peptides present as uncleaved extension chains containing an amino acid composition different from the alpha chain component. The investigators concluded, however, that the overall similarities in amino acid composition and in the types of cyanogen bromide cleavage peptides indicate that the fibers of the central and posterior peripheral regions of the vitreous are composed of a collagen that should be classified as type II. Schmut and associates²³ used differential salt precipitation of pepsin-solubilized collagen from bovine vitreous and found that type II collagen is the major component of native vitreous fibers. Linsenmayer and collaborators²⁴ measured in vivo synthesis of types I and II collagen in chick embryo vitreous by radioimmunoprecipitation after tritiated proline labeling and found that over 90% of the labeled material in the vitreous was type II collagen. Snowden²⁵ provided further physicochemical evidence in support of the similarities between vitreous and cartilage collagens. Considered as a whole, this body of work may explain why certain clinical phenomena, such as inborn errors of type II collagen metabolism in arthro-ophthalmopathies, manifest phenotypic expression in each of these two tissues.

There are, however, distinct differences in the chemical composition of vitreous and cartilage collagens that are only partly due to the presence of terminal peptide constituents in vitreous collagen. Swann and Sotman²⁶ have demonstrated that the carbohydrate content of pepsin-solubilized vitreous α chains is significantly greater than cartilage α chains, indicating that the carbohydrate side chains of vitreous collagen are largely composed of disaccharide units similar to those found in basement membrane collagen. They proposed that these distinct chemical features are related to the special structure of the mature vitreous fibrils in vivo. Liang and Chakrabarti²⁷ have shown that there are differences between bovine cartilage and vitreous with respect to collagen fibril growth, melting temperature, and fluorescence with a hydrophobic fluorescent probe. These investigators and others²⁸ proposed that vitreous collagen should be considered a special type II collagen. Ayad and Weiss²⁹ demonstrated that type II is the major vitreous collagen in bovine vitreous but collagens composed of α1, α2, and α3 chains as well as C-PS disulfide-bonded collagen were present in concentrations similar to those in cartilage. In contrast to cartilage, however, vitreous type II collagen was significantly more hydroxylated in the lysine and proline residues. The α1, α2, and α3 collagen chains were interpreted by Van der Rest³⁰ to represent type IX collagen. Eyre and colleagues³¹ felt that there was evidence to indicate the presence of type V collagen in vitreous. Furthermore, with respect to the disulfide-bonded collagen, vitreous had three times more C-PS1 and C-PS2 collagens than cartilage although the molar ratio of C-PS1 to C-PS2 in both was 1:1, suggesting that in both tissues these collagens are components of a larger molecule, possibly a derivative of type IX collagen. In this regard vitreous is once again similar to cartilage, insofar as both contain type IX collagen, although the two tissues differ in the sizes of type IX collagen chains.³² Hong and Davison³³ have identified a procollagen in the soluble fraction of rabbit vitreous that was identified as type II by segment-long spacing-banding patterns. Detection of a propeptide extension only at the N-terminus prompted these investigators to conclude that this was a novel type II procollagen. Such distinctive characteristics are possibly related to the unique physiologic roles of vitreous, in particular, its mechanical function.¹²

Vitreous collagen fibrils are organized as a triple helix of three alpha chains (Fig. 2). The collagen fibrils of vitreous are heterotypic, consisting of more than one collagen type. Recent studies of pepsinized forms of collagen confirm that vitreous contains collagen type II, a hybrid of types V/XI, and type IX ^34,35

TYPE II COLLAGEN.

Type II collagen, a homotrimer composed of three identical α chains designated as [α1 (II)]₃, comprises 75% of the total collagen content in vitreous. When first synthesized as a procollagen and secreted into the extracellular space, type II collagen is highly soluble. The activity of N-proteinase and C-proteinase enzymes reduces the solubility and enables type II collagen molecules to cross-link covalently in a quarter-staggered array. Within this array are likely to be N-propeptides, which probably extend outward from the surface of the forming fibril.³⁵ This may influence the interaction of the collagen fibril with other components of the extracellular matrix. Recent studies³⁶ combined immunolocalization with Western blot analysis of macromolecules extracted from bovine vitreous collagen fibrils and found that the pN-type IIA procollagen is located on the surface of the vitreous collagen fibril. The finding³⁷ that type IIA procollagen propeptides specifically bind transforming growth factor (TGF)-β₁ and bone morphogenic protein-2 (BMP-2) supports the concept that in certain circumstances, such growth factors and cytokines interact with vitreous fibrils to promote the cell migration and proliferation that result in proliferative diabetic retinopathy and proliferative vitreoretinopathy.

TYPE IX COLLAGEN.

Type IX collagen is a heterotrimer which is disulfide-bonded with an [α 1 (IX) α 2 (IX) α 3 (IX)] configuration. This heterotrimer is orientated regularly along the surfaces of the major collagen fibrils in a D periodic distribution, where it is cross-linked onto the fibril surface. Type IX is not a typical collagen but is a member of the fibrillar-associated collagens with interrupted triple helixes (FACIT) group of collagens. It contains collagenous regions described as COL1, COL2, and COL3 interspersed between noncollagenous regions called NC1, NC2, NC3, and NC4.^35,38 In vitreous, as opposed to cartilage, the NC4 domain is small and, therefore, not highly charged and not likely to exhibit extensive interaction with other extracellular matrix components.³⁹ In vitreous, type IX collagen always contains a chondroitin sulfate glycosaminoglycan chain,^35,38 which is linked covalently to the α 2 (IX) chain at the NC3 domain; this enables the molecule to assume a proteoglycan form. Electron microscopy of vitreous stained with cationic dyes enables visualization of the chondroitin sulfate chains of type IX collagen. In some of these studies, sulfated glycosaminoglycans are found distributed regularly along the surface of vitreous collagen fibrils,⁴⁰ and often bridge between neighboring collagen fibrils. Duplexing of glycosaminoglycan chains from adjacent collagen fibrils may result in a ladderlike configuration.⁴¹ However, this hypothesis of vitreous organization has yet to be confirmed.

TYPE V/XI COLLAGEN.

Ten percent of vitreous collagen is a hybrid V/XI collagen, which is believed to comprise the central core of the major collagen fibrils of vitreous.⁴² Type V/XI is a heterotrimer that contains α 1 (XI) and α 2 (V) in two chains, while the nature of the third chain is presently not known. Along with type II collagen, type V/XI is a fibril-forming collagen. While the interaction of the fibril with other extracellular matrix components is probably influenced by a retained N-propeptide which protrudes from the surface of the fibril in cartilage,⁴² it is not known whether this is the case in vitreous.

TYPE VI COLLAGEN.

Although there are only small amounts of type VI collagen in vitreous, the ability of this molecule to bind both type II collagen and HA suggests that it could be important in organizing and maintaining the supramolecular structure of vitreous gel. Aggregates of type VI collagen have been found in the cortical vitreous of a patient with macular hole,⁴³ but it is not clear whether this plays any role at all in the pathogenesis of this condition.

GLYCOSAMINOGLYCANS

GAGs are polysaccharides of repeating disaccharide units, each consisting of hexosamine (usually N-acetyl glucosamine or N-acetyl galactosamine) glycosidically-linked to either uronic (glucuronic or iduronic) acid or galactose. The nature of the predominant repeating unit is characteristic for each glycosaminoglycan and the relative amount, molecular size, and type of glycosaminoglycan are said to be tissue-specific.⁴⁴ GAGs do not normally occur as free polymers in vivo but are covalently linked to a protein core, the ensemble called a proteoglycan. A sulfated group is attached to oxygen or nitrogen in all GAGs except HA. Balazs⁴⁵ first documented the presence of sulfated galactosamine-containing GAGs in bovine vitreous (less than 5% of total vitreous glycosaminoglycans), and others^46,47 identified these as chondroitin-4-sulfate and undersulfated heparan sulfate. Studies in the rabbit⁴⁸ found a total vitreous glycosaminoglycans content of 58 ng with 13% chondroitin sulfate and 0.5% heparan sulfate.

HYALURONAN.

Although HA is present throughout the body, it was first isolated from bovine vitreous by Meyer and Palmer in 1934.⁴⁹ In humans, HA first appears after birth and then becomes the major vitreous GAG. It has been proposed that hyalocytes synthesize HA, but other plausible candidates are the ciliary body and retinal Müller cells. Whereas the synthesis of HA seems to continue at a constant rate in the adult without extracellular degradation, HA levels are in a steady-state because the molecule escapes via the anterior segment of the eye.⁵⁰ Laurent and Fraser⁵⁰ showed that the passage of HA from the vitreous to the anterior segment is strongly molecular-weight–dependent, indicating a diffusion-controlled process. In contrast, disappearance of HA from the anterior chamber is independent of molecular weight, suggesting that this is controlled by bulk flow.

HA is a long, unbranched polymer of repeating glucuronic acid β-1,3-N,N-acetylglucosamine disaccharide moieties linked by β-1–4 bonds. It is a linear, left-handed, threefold helix⁵¹ with a rise per disaccharide on the helix axis of 0.98 nm (Fig. 3).⁵² Rotary shadowing electron microscopy of human and bovine vitreous detected lateral aggregates of HA that form an anastomosing three-dimensional network.⁵² This periodicity, however, can vary depending on whether the helix is in a compressed or extended configuration.⁵³ Changes in the degree of extension of HA could be important in the role vitreous plays in retinal disease. Indeed, the volume of the unhydrated HA molecule is approximately 0.66 cm³/g, whereas the hydrated specific volume is 2,000 to 3,000 cm³/g.⁵³ Thus, the degree of hydration has a significant influence on the size and configuration of the HA molecular network. Although there is no definitive evidence that adjacent HA chains bind to one another, Brewton and Mayne⁵⁴ first proposed such an arrangement. Recent rotary shadowing electron microscopy studies⁵⁵ of bovine and human vitreous found lateral aggregates of HA that formed three-dimensional latticelike networks. HA also interacts with the surrounding mobile ions and can undergo changes in its conformation that are induced by changes in the surrounding ionic milieu.⁵⁶ A decrease in surrounding ionic strength can cause the anionic charges on the polysaccharide backbone to repel one another, resulting in an extended configuration of the macromolecule. An increase can cause contraction of the molecule and, in turn, the entire vitreous body. As a result of HA's entanglement and immobilization within the vitreous collagen fibril matrix, this mechanical force can be transmitted by collagen fibrils to the retina, optic disc, and other structures, such as neovascular complexes. In this way, changes in the ionic milieu of vitreous may be converted into mechanical energy via extension or contraction of the HA macromolecule. This can be important in certain pathologic conditions that feature fluctuations in ionic balance and hydration, such as diabetes.⁵⁷

The sodium salt of HA has a molecular weight of 3 to 4.5 × 10⁶ in normal human vitreous.⁴⁵ Laurent and Granath⁵⁸ used gel chromatography and found the average molecular weight of rabbit vitreous to be 2 to 3 × 10⁶ and of bovine vitreous to be 0.5 to 0.8 × 10⁶. In these studies there were age-related differences in the bovine vitreous, in which HA molecular weight varied from 3 × 10⁶ in the newborn calf to 0.5 × 10⁶ in old cattle. Furthermore, there may be several species of HA within vitreous that have polysaccharide chains of different lengths⁵⁹ with a variable distribution in different topographic regions within the vitreous body.⁶⁰

An important property of HA is that of steric exclusion.⁶¹ With its flexible linear chains and random coil conformation, HA occupies a large volume and resists the penetration of this volume by other molecules to a degree dependent upon their size and shape.⁵³ This excluded volume effect can influence equilibria between different conformational states of macromolecules and alter the compactness or extension of these molecules. Steric exclusion also causes an excess of osmotic pressure when such compounds as albumin and HA are mixed, because the resultant osmotic pressure is greater than the sum of the two components. This could be important in diabetes, in which vascular incompetence can increase vitreous concentrations of serum proteins such as albumin. These osmotic effects can induce contraction and expansion of the vitreous body similar to the foregoing description of hydration and ion-induced changes within vitreous, and can similarly play an important role in neovascularization and diabetic vitreous hemorrhage.⁵⁷ An increase in the chemical activity of a compound because of steric exclusion can cause its precipitation if the solubility limit is reached. This could be important in the formation of pathologic vitreous opacities, such as asteroid hyalosis and amyloidosis (see below).

CHONDROITIN SULFATE.

Vitreous contains two types of chondroitin sulfate proteoglycans. The minor type is actually type IX collagen, which is described above. The majority of vitreous chondroitin sulfate, however, is in the form of versican.⁵⁵ This large proteoglycans (molecular weight [MW] = 380 kd) has a globular N-terminal which binds HA via a 45-kd link protein. Thus, in human but not bovine vitreous, versican is believed to form complexes with HA as well as microfibrillar proteins, such as fibulin-1 and fibullin-2.⁶² The concentration of versican in the human vitreous is 0.06 mg/mL of vitreous gel, representing approximately 5% of the total protein content. Each versican molecule has 5 to 7 chondroitin sulfate side chains (MW = 37 kd each), which form aggregates with vitreous HA. It also binds lectins via the C-terminal region. Binding by both the C- and N-terminal domains is believed to be the manner by which versican stabilizes the macromolecular structures and organization of vitreous.⁶³

HEPARAN SULFATE.

This sulfated proteoglycans is normally found in basement membranes and on cell surfaces throughout the body. It was first detected in bovine vitreous in 1977,⁶⁴ and in chick vitreous (as agrin) in 1995.⁶⁵ However, it is not clear whether heparan sulfate is a true component of vitreous or a contaminant from adjacent basement membranes, such as the internal limiting lamina of the retina.⁶⁴ As pointed out by Bishop⁶² this may also be the case for nodogen-1, the aforementioned fibulins, and fibronectin.

Noncollagenous Structural Proteins

FIBRILLINS.

Fibrillin-containing microfibrils are more abundant in vitreous than the type VI collagen microfibrils described above. They are the found in vitreous gel as well as in the zonules of the lens. This fact explains why in Marfan's syndrome the defects in the gene encoding fibrillin-1 (FBN1 on chromosome 15q21) result in both ectopia lentis and vitreous liquefaction.⁶² The latter probably plays a role in the frequent occurrence of rhegmatogenous retinal detachment in these patients.

OPTICIN.

The major noncollagenous protein of vitreous is a leucine-rich repeat (LRR) protein, which is bound to the surface of the heterotypic collagen fibrils, known as opticin.^67–69 Formerly called vitrican, opticin is believed to be important in collagen fibril assembly and in preventing the aggregation of adjacent collagen fibrils into bundles. Thus, a breakdown in this property or activity may play a role in age-related vitreous degeneration (see below).⁷⁰

VIT1.

Another novel vitreous protein is VIT1, a collagen-binding macromolecule.⁷¹ Because of its propensity to bind collagen, this highly basic protein may play an important role in maintaining vitreous gel structure.

Glycoproteins

Glycoproteins are macromolecules of the ground substance existing as heteropolysaccharides rather than homogeneous repeating disaccharide units, such as found in GAGs. They are mostly proteinaceous and contain only a minor carbohydrate component (5% to 10% by weight). According to Balazs,⁷² the most important difference between vitreous and serum proteins is the high content of glycoproteins in vitreous, because these constitute 20% of the total noncollagenous protein content of vitreous.

There are two types of vitreous glycoproteins: those found associated with collagen fibrils and those that freely diffuse in the interfibrillar space. Although some of the soluble glycoproteins enter vitreous from serum, many are believed to be specific to vitreous. Studies using the radiolabeled precursor ³H-fucose detected 14 fractions of labeled polypeptides.⁷³ The same results were obtained using tritiated amino acids,⁷⁴ suggesting that more than one component is synthesized within the eye, probably at the ciliary body. Rhodes and co-workers⁷⁵ studied the incorporation of tritiated-fucose into rabbit vitreous and surrounding tissues, concluding that there is continuous renewal of the glycoproteins in and around the vitreous body. They suggested that this process could serve as an index of metabolic activity in normal and pathologic states. Hageman and Johnson⁷⁶ documented the distribution of vitreous glycoproteins in several species, including man, and characterized these glycoproteins based on differential staining to various fluorescein isothiocyanate-conjugated lectins.

There is no clear understanding of the role of glycoproteins in vitreous. Through Western blotting techniques, an acidic glycoprotein has been identified that is unique to cartilage and vitreous.⁷⁷ It is known as cartilage oligomeric matrix protein and has a characteristic five-armed structure. The function of this molecule in vitreous is unknown. Nevertheless, it represents more evidence linking vitreous with joints and may explain why both structures are often simultaneously involved in certain syndromes. Recent findings, reviewed by Bishop et al,⁷⁸ have led to the concept that glycoproteins and other connective tissue macromolecules, as well as basement membrane macromolecules that are synthesized by the ciliary body are important in the biosynthesis of vitreous as well as the internal limiting lamina of the retina. The former has significant implications for development of the eye as well.

Miscellaneous Components

ANTIOXIDANTS.

High performance liquid chromatography measurements of vitreous ascorbic acid concentration showed values approximating 0.43 mmol/kg, which represent a vitreous to plasma ratio of 9:1.⁷⁹ Vitreous levels this much higher than plasma concentrations are believed to be caused by active transport by the ciliary body epithelium. Such high vitreous concentrations of ascorbic acid may relate to the ability of this compound to absorb ultraviolet light⁸⁰ and serve as a free-radical scavenger.⁸¹ This would protect the retina and lens from the untoward effects of metabolic and light-induced singlet oxygen generation. Indeed, a study in an animal model showed that the light-induced free radicals induced vitreous liquefaction.⁸² Ascorbic acid may also protect against oxidative damage due to inflammation.⁸³ Vitreous levels of serum-derived ceruloplasmin and transferrin also increase after experimental inflammation,⁸⁴ presumably to provide additional antioxidant protection.

SUPRAMOLECULAR ORGANIZATION

As described by Mayne,⁸⁵ vitreous is organized as a dilute meshwork of collagen fibrils interspersed with extensive arrays of long HA molecules. The collagen fibrils provide a scaffold-like structure that is inflated by the hydrophilic HA. If collagen is removed, the remaining HA forms a viscous solution; if HA is removed, the gel shrinks⁵⁶ but is not destroyed. Early physiologic observations⁸⁶ suggested the existence of an interaction between HA and collagen which stabilizes collagen. Gelman and associates⁸⁷ have shown that several GAGs, including HA, stabilized the helical structure of collagen, so that the melting temperature of collagen was increased from 38° C to 46°C. Hyaluronidase decreased the thermal stability of cartilage collagen and the addition of chondroitin-6-sulfate to hyaluronidase-treated collagen in turn increased the thermal stability. Snowden⁸⁸ demonstrated that the shrinkage temperature of tendon collagen is dependent linearly on the concentration of chondroitin sulfate in the surrounding fluid. Biomechanical studies⁸⁹ of vitreous viscoelasticity suggested that collagen similarly affects HA. The exact nature of the interaction between vitreous macromolecules that results in the aforementioned phenomena has yet to be determined, although there are several possibilities: interactions on a chemical or electrostatic level, or via an intermediary molecule. Concerning a chemical interaction, Balazs⁴⁵ hypothesized that the hydroxylysine amino acids of collagen mediate polysaccharide binding to the collagen chain via O-glycosidic linkages. These polar amino acids are present in clusters along the collagen molecule, which may explain why proteoglycans attach to collagen in a periodic pattern.⁴⁰

Mathews⁹⁰ observed the reversible formation of complexes of an electrostatic nature between solubilized collagen and various GAG. He suggested that collagen-HA interaction occurs on a physicochemical rather than chemical level. Podrazky and co-workers⁹¹ demonstrated that the sulfate group of a GAG was largely responsible for interactions with the guanidine groups of arginine and ε-amino groups of lysine in collagen. When these highly charged groups on the collagen fibrils are bound by advanced glycation end products, such as occurs in diabetes⁵⁷ and aging,⁹² destruction of normal vitreous structure results, leading ultimately to diabetic,⁹³ age-related,^94,95 structural changes. Others⁹¹ demonstrated that the sulfate group of a GAG was largely responsible for such interactions with the guanidino groups of arginine and epsilon-amino groups of lysine in collagen. Comper and Laurent⁵⁶ proposed that in vitreous, electrostatic binding occurs between negatively charged polysaccharides and positively charged proteins. These authors extensively reviewed the existing data characterizing the electrostatic properties of glycosaminoglycans and the factors influencing their electrostatic interactions with different ions and molecules.

HA-collagen interaction in vitreous may be mediated by a third molecule.⁹⁶ Studies by Hong and Davison⁹⁷ have identified a type II procollagen in the soluble fraction of rabbit vitreous and raised the question of a possible role for this molecule in mediating collagen-HA interaction. Measurements of the dynamic viscoelasticity of bovine vitreous showed that the shapes of the master relaxation curves of the vitreous body are similar to those of lightly cross-linked polymer systems.⁹⁸ Notably, the behavior of these relaxation curves is different from those observed in solutions of HA and collagen. This suggests that the physicochemical properties of vitreous in vivo are not simply the result of a combination of these two molecular elements, but that HA and collagen form a lightly cross-linked polymer system. Swann and colleagues⁹⁹ have demonstrated large amounts of noncollagenous protein associated with collagen in the insoluble residue fraction of vitreous. Asakura⁴⁰ studied bovine vitreous by ruthenium red staining and demonstrated the presence of amorphous structures on collagen fibrils at 55- to 60-nm intervals along the fibrils that are believed to be HA (Fig. 4). Filaments connect the collagen fibrils and these amorphous masses. These filaments may represent link structures that are either proteoglycans or a glycoprotein, such as hyaluronectin.¹⁰⁰ In cartilage, link glycoproteins have been identified that interact with proteoglycans.¹⁰¹ Supramolecular complexes of these glycoproteins are thought to occupy the interfibrillar spaces in cartilage and may have a similar role in vitreous. In the cornea, chondroitin sulfate, and keratan sulfate bridge the interfibrillar spaces and keep the fibrils at specified distances to achieve transparency.¹⁰² This is clearly important in vitreous as well, where HA could bind to collagen fibrils by such linkage molecules, most probably in a repeating order. This type of arrangement would bind collagen fibrils to the protein core of a proteoglycans, such as chondroitin sulfate, as described by Asakura,⁴⁰ keratan sulfate, or both, as in the cornea, and would organize the network in a manner to keep the vitreous collagen fibrils apart by at least one wavelength of incident light, the critical distance needed to minimize light scattering. Although such supramolecular organization to maintain vitreous transparency was proposed⁴¹ more than a decade ago, no evidence has yet been generated.

Bishop⁶² has proposed that to understand how vitreous gel is organized and stabilized requires an understanding of what prevents collagen fibrils from aggregating and by what means the collagen fibrils are connected to maintain a stable gel structure. Scott and colleagues¹⁰³ have proposed that the chondroitin sulfate chains of type IX collagen bridge between adjacent collagen fibrils in a ladderlike configuration spacing them apart. This arrangement might account for vitreous transparency, because keeping vitreous collagen fibrils separated by at least one wavelength of incident light would minimize light scattering, allowing unhindered transmission of light to the retina for photoreception. However, depolymerizing with chondroitinases does not destroy the gel, suggesting that chondroitin sulfate side chains are not essential for vitreous collagen spacing. Complexed with HA, however, the chondroitin sulfate side chains might space apart the collagen fibrils sufficiently.^41,103 Yet, Bishop believes that this form of collagen-HA interaction would only be very weak. Instead, he proposes that the leucine-rich repeat (LRR) protein opticin is the predominant structural protein in short-range spacing of collagen fibrils. Concerning long-range spacing, Scott⁴¹ and Mayne et al¹⁰⁴ have claimed that HA plays a pivotal role in stabilizing the vitreous gel via this mechanism. However, studies¹⁰⁵ using HA lyase to digest vitreous HA demonstrated that the gel structure was not destroyed, suggesting that HA is not essential for the maintenance of vitreous gel stability and leading to the proposal that collagen alone is responsible for the gel state of vitreous.

Several types of collagen-HA interactions may occur in different circumstances. Further investigation must be undertaken to identify the nature of collagen-HA interaction in vitreous. This question is important for an understanding of normal vitreous anatomy and physiology but also as a means by which to understand the biochemical basis for age- and disease-related vitreous liquefaction and posterior vitreous detachment (PVD).

SPECIES VARIATIONS

Vitreous of all species is composed of essentially the same extracellular matrix elements, organized to fill the center of the eye with a clear viscous substance surrounded by a dense cortex that is attached to the basal laminae of surrounding cells. There are, however, species variations in the relative concentrations of the major structural components (i.e., HA and collagen). These differences explain variations in the rheologic (gel–liquid) state of vitreous in different species. It should be emphasized that there are also age-related differences in higher order species. Consequently, when selecting an appropriate animal with which to model human diseases for scientific investigation, consideration must be given to these species variations and age-related differences.

Significant species differences exist for HA, which is present in vitreous of all species studied except fish. In fish, the posterior and peripheral vitreous adjacent to the retina is a gel. The anterior and central vitreous is liquid. Because there is no anterior vitreous cortex, this liquid vitreous is continuous with the posterior and anterior chambers, thereby surrounding the lens. In place of HA, the fish vitreous contains icthyosan, a large (MW = 2 to 4 × 10⁶) nonsulfated polysaccharide consisting of HA and a chondroitin chain held together by noncolvent bonds.¹⁰⁶ HA concentration in human vitreous (approximately 192 μg/mL) is about the same as in the rhesus monkey, which is less than in the owl monkey (291.8 ± 18.8 μg/mL) and bovine (469 ± 44.0 μg/mL) vitreous.^107,108 The molecular weight of rhesus monkey HA is 2.9 ± 0.06 × 10⁶,¹⁰⁸ which is significantly less than in humans (4.6 × 10⁶; p > 0.001).¹⁰⁷ In pigs, both collagen and HA are found in half the concentration as in humans.

Collagen concentration in vitreous gel is estimated to be approximately 60 μg/mL in bovine eyes and approximately 300 μg/mL in human eyes.¹⁰⁹ Snowden and Swann¹¹⁰ demonstrated that collagen fibrils in rabbit vitreous measure 7 nm in diameter, whereas bovine and canine vitreous collagen fibrils are between 10 and 13 nm in diameter. Human vitreous collagen fibrils are 10 to 25 nm in diameter.¹¹¹ Although the baby owl monkey vitreous is a gel with a network of collagen-like fibrils that have a diameter of 11.3 nm, the adult owl monkey has no collagen and vitreous is a viscous fluid that contains mostly high molecular weight HA.¹¹²

EMBRYOLOGY

Interfaces

During invagination of the optic vesicle, the basal lamina of the surface ectoderm enters the invagination along with the ectodermal cells that have become specialized neural ectoderm.¹¹³The ectodermal cells that are on the surface of the inner with the outer basal lamina give rise to retinal pigment epithelium attached to Bruch's membrane, while the invaginating neural ectoderm and its basal lamina give rise to the neural retinal cells adherent to the internal limiting lamina (ILL). Thus, the basal laminae of both the retina and RPE have the same embryologic origin. Figure 5 demonstrates the continuity of these two basal laminae. It is important to appreciate that these basal laminae serve as interfaces between adjacent ocular structures. In the case of the ILL, this basal lamina is the interface between the retina and vitreous. Bruch's membrane separates the RPE and retina from the choroid (neural crest origin).

These interfaces play an important role in a significant biologic event that underlies one of the most devastating causes of blindness in humans: neovascularization. At the ILL interface between vitreous and retina, neovascularization in advanced diabetic retinopathy and other ischemic retinopathies, including retinopathy of prematurity (see below), is a significant cause of vision loss. At the level of Bruch's membrane, an interface of identical embryologic origin as the ILL, neovascularization in age-related macular degeneration is a significant and growing problem. Both of these conditions result from vascular endothelial cell migration and proliferation onto and into interfaces of the same embryologic origin: the basal lamina of the surface ectoderm. Improving our understanding of endothelial cell interaction with these interfaces should provide new insights into therapy and prevention of these important disorders.

Vitreous Body

Early in embryogenesis, the vitreous body is filled with blood vessels known as the vasa hyaloidea propia. This network of vessels arises from the hyaloid artery, which is directly connected to the central retinal artery at the optic disc. The vessels branch many times within the vitreous body and anastomose anteriorly with a network of vessels surrounding the lens, the tunica vasculosa lentis. This embryonic vascular system attains its maximum prominence during the ninth week of gestation or 40-mm stage.¹¹⁴ Atrophy of the vessels begins posteriorly with dropout of the vasa hyaloidea propria, followed by the tunica vasculosa lentis. At the 240-mm stage (seventh month) in the human, blood flow in the hyaloid artery ceases.¹¹⁵ Regression of the vessel itself begins with glycogen and lipid deposition in the endothelial cells and pericytes of the hyaloid vessels.¹¹⁵ Endothelial cell processes then fill the lumen and macrophages form a plug that occludes the vessel. The cells in the vessel wall then undergo necrosis and are phagocytized by mononuclear phagocytes.¹¹⁶ Gloor¹¹⁷ claims that macrophages are not involved in vessel regression within the embryonic vitreous but that autolytic vacuoles form in the cells of the vessel walls, perhaps in response to hyperoxia. Interestingly, the sequence of cell disappearance from the primary vitreous begins with endothelial and smooth muscle cells of the vessel walls, followed by adventitial fibroblasts and lastly phagocytes,¹¹⁸ consistent with a gradient of decreasing oxygen tension.

It is not known precisely what stimulates regression of the hyaloid vascular system, but studies have identified a protein native to the vitreous that inhibits angiogenesis in various experimental models.^119–121 Teleologically, such activity seems necessary if a transparent tissue is to inhibit cell migration and proliferation and minimize light scattering to maintain transparency. This may also be the mechanism that induces regression of the vasa hyaloidea propia. Thus, activation of this protein and its effect on the primary vitreous may be responsible for the regression of the embryonic hyaloid vascular system as well as the inhibition of pathologic neovascularization in the adult. Hyaloid vessel regression may also result from a shift in the balance between growth factors promoting new vessels, such as vascular endothelial growth factor (VEGF)-A, and those inducing regression, such as placental growth factor.¹²²

Recent studies^123,124have suggested that vasa hyaoidea propria and the tunica vasculosa lentis regress via apoptosis. Mitchell and colleagues¹²³ pointed out that the first event in hyaloid vasculature regression is endothelial cell apoptosis and propose that lens development separates the fetal vasculature from VEGF-producing cells, decreasing the levels of this survival factor for vascular endothelium, inducing apoptosis. After endothelial cell apoptosis, there is loss of capillary integrity, leakage of erythrocytes into the vitreous, and phagocytosis of apoptotic endothelium by macrophages, which were felt to be important in this process. Subsequent studies by a different group¹²⁵ confirmed the importance of macrophages in promoting regression of the fetal vitreous vasculature and further characterized these macrophages as hyalocytes. Meeson and colleagues¹²⁶ proposed that there are actually two forms of apoptosis that are important in regression of the fetal vitreous vasculature. The first (initiating apoptosis) results from macrophage induction of apoptosis in a single endothelial cell of an otherwise healthy capillary segment with normal blood flow. The isolated dying endothelial cells project into the capillary lumen and interfere with blood flow. This stimulates synchronous apoptosis of downstream endothelial cells (secondary apoptosis) and ultimately obliteration of the vasculature. Removal of the apoptotic vessels is achieved by hyalocytes.

A better understanding of this phenomenon may provide insights into new ways to induce the regression of pathologic angiogenesis or inhibit neovascularization in such conditions as proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). Indeed, the recently developed synthetic VEGF inhibitors seem to be of limited usefulness in treating pathologic neovascularization in exudative AMD and this or a superior inhibitory mechanism may prove to be useful in other proliferative retinopathies, such as retinopathy of prematurity.

Retinopathy of Prematurity

The pathogenesis of retinopathy of prematurity (ROP) begins with birth prior to complete maturation and development of the peripheral retina. Postnatal oxygen administration triggers retinal vasoconstriction with endothelial cell necrosis and vaso-obliteration in response to hyperoxia.^127–129 After the discontinuation of supplemental oxygen, arterial pO₂ levels return to normal and the obliterated (or at best, highly constricted) vessels are not adequately reperfused, causing the peripheral retina they subserve to become ischemic and release neovascular growth factors. An alternative hypothesis of pathogenesis proposes that spindle cells in the immature peripheral retina are stimulated by excessive amounts of reactive oxygen species, whether related to oxygen therapy and subsequent relative hypoxia or other metabolic circumstances, to release angiogenic growth factors.¹³⁰ In either case, the result is migration and proliferation of capillary endothelial cells that form new blood vessels at the posterior ridge of tissue between the vascularized and avascular retina. This results in neovascularization arising from the ridge that demarcates the developed posterior retina from the immature peripheral retina (Fig. 6). The new vessels grow into the vitreous body, onto and into the plane that demarcates the interface between the gel vitreous posteriorly (overlying developed retina) and the liquid vitreous anteriorly (overlying immature retina). While somewhat similar to neovascularization in diabetic retinopathy,¹³¹ the new vessels in ROP grow farther anteriorly and with greater exuberance. This is perhaps because of the participation of cells of the ocular fetal vasculature, whose apoptosis has been retarded or arrested by the presence of high levels of VEGF.¹³²

There are no clearly identified vitreous changes during stages I and II of acute ROP, although this may simply be due to our present inability to detect such abnormalities.¹³³ Indeed, the abundance of reactive oxygen species in the retina and vitreous of premature infants could induce widespread vitreous liquefaction.⁸² There are also likely to be localized areas of liquid vitreous, particularly at the periphery. At surgery for stage IV-A ROP with retinal detachment, there is a trough in the periphery.¹³⁴ This structure is most likely the consequence of underlying immature retina in the periphery, with consequent lack of typical gel vitreous synthesis, normally a Müller cell function, overlying the immature retina. The liquid vitreous trough is probably present early in the natural history of disease but goes undetected by present vitreous imaging techniques.¹³³ Such disruption of normal vitreous composition and structure probably alters a number of physiologic processes within the vitreous, including the ability of vitreous to inhibit cellular and vascular invasion.¹³⁵ Furthermore, the interface between posterior gel vitreous and peripheral liquid vitreous at the ridge causes vitreous traction to be exerted at the retinal ridge.

In stage III ROP, new blood vessels extend from the inner retina into the vitreous cortex. The cortex, overlying the rear guard of differentiated capillary endothelial cells, becomes opaque and contains linear, fibrous structures adjacent to a large pocket of liquid vitreous.¹³⁶ In advancing from stage III to stage IV, the neovascular tissue arising from the rear guard grows through the vitreous body toward Wiegert's ligament on the posterior lens capsule.¹³⁶ This configuration of neovascularization is probably the result of cell migration and proliferation along the walls of the future Cloquet's canal or the tractus hyaloideus of Eisner. Cells of the primary vitreous likely contribute to the formation of the dense central vitreous stalk and retrolental membrane seen in the cicatricial stage, because these cells could also undergo migratory and proliferative responses to intraocular angiogenic stimuli.

Familial Exudative Vitreoretinopathy

This autosomal (long arm of chromosome 11) dominant (sporadic cases of X-linked recessive disease have been reported) vitreoretinopathy features inner retinal non-perfusion, neovascularization and retinal detachment. The histologic vascular changes are indistinguishable from those of ROP, however, patients with familial exudative vitreoretinopathy (FEVR) have no history of prematurity or neonatal oxygen exposure.^137,138 Mild forms of the disease are characterized by areas of peripheral nonperfusion, cystoid degeneration, and condensation of the temporal vitreous base and vitreous membranes. Moderate forms involve vitreous base traction, flat or elevated neovascularization, and retinal or subretinal exudates. The severe forms of FEVR, which usually cause vision loss, are similar to cicatricial ROP, featuring iris atrophy, neovascular glaucoma, cataracts, falciform retinal folds, macular heterotopia, and retinal detachment.^139,140 In a recent series¹⁴¹ of 28 patients who underwent vitreoretinal surgery, 25 had rhegmatogenous and 2 had traction retinal detachments. Vitreoretinal adhesion was so strong in the avascular peripheral retina that iatrogenic retinal breaks occurred in 22 of 28 eyes during surgery. It is quite plausible that in similar fashion to ROP, FEVR has poorly developed vitreous overlying the nonperfused, and therefore immature, peripheral retina. The instability between the well-developed posterior gel vitreous and the liquefied peripheral anterior vitreous (Fig. 6) probably exerts traction upon the retina inducing the rhegmatogenous and tractional detachments.

POSTNATAL DEVELOPMENT

Vitreous Size

Vitreous undergoes significant growth during childhood. A study of 926 children found that the average length of the newborn vitreous is 10.5 mm in males and 10.2 mm in females.¹⁴² Immediate postnatal development was studied in the rhesus monkey where axial length was found to increase most rapidly during the first 5 to 7 months, increase at a slower pace during the next 6 months, and continue to increase slowly until 4 years of age. This purportedly corresponds closely to axial elongation in humans.¹⁴³ Fledelius¹⁴⁴ used ultrasound to measure ocular dimensions in adolescents and found that in the absence of changes in refractive status, there was a mean vitreous elongation of 0.35 mm from age 10 to 18 years. By the age of 13 years, the axial length of the human vitreous body increases to 16.1 mm in the male and 15.6 mm in the female.¹⁴⁴ Derangements in ocular growth that result in ametropia are associated with abnormal vitreous dimensions. In children born prematurely (birth weight less than 2000 g), axial lengths of the eye and vitreous at age 10 years are less than normal, a deficiency that is also present at the age of 18 years, suggesting that prematurity does not simply induce a temporary delay of eye growth but a permanent deficit.¹⁴⁴

Vitreous Biochemistry

Both collagen and HA are synthesized during development to adulthood. The synthesis of collagen only keeps pace with increasing vitreous volume during growth. Thus, the overall concentration of collagen within vitreous is unchanged during this period (Fig. 7). Total collagen content in the gel vitreous decreases during the first few years of life and then remains at approximately 0.15 mg until the third decade. Because collagen concentration does not appreciably increase during this time when the size of the vitreous increases, the density of collagen fibrils decreases. This may weaken the collagen network and destabilize the gel. Because there is net synthesis of HA during this time (Fig. 8), the thinning collagen network is stabilized (see above).

Vitreous Body

When secondary vitreous formation occurs normally, the result is a clear, viscoelastic gel that fills the center of the eye (Fig. 1). Because of the intricate interaction between HA and collagen, the vitreous body is transparent centrally, with a dense periphery known as the vitreous cortex. At times there is a visible central structure, known as Cloquet's canal, which is the site of the former hyaloid artery (Figs. 9B and 10). The solid vitreous body of youth scatters little or no incident light, remaining relatively clear through the first few decades of life (Fig. 11) until changes occur with aging, initially on a molecular level and ultimately on a macroscopic structural level that affects the entire vitreous.

Anomalous Vitreous Development

Proper vitreous biosynthesis during embryogenesis depends on normal retinal development because at least some of the structural components in vitreous are synthesized by retinal Müller cells.¹⁴⁵ A clear gel, typical of normal secondary vitreous, appears only over normally developed retina. Thus, in developmental anomalies such as retinopathy of prematurity and familial exudative vitreoretinopathy, the vitreous overlying undeveloped retina in the peripheral fundus is a viscous liquid and not a gel. How far this finding extends posteriorly in retinopathy of prematurity depends upon the gestational age at birth. Because the retina develops anteriorly from the optic disc, the more immature the individual, the less developed the retina in the periphery, especially temporally, and the more vitreous structure is liquid and not gel. Inborn errors of type II collagen metabolism, such as in the arthro-ophthalmopathies, often feature very liquefied vitreous early in life and these individuals are at high risk of rhegmatogenous retinal detachment. It is also possible that the liquid vitreous observed in myopia is similarly caused by myopia-related abnormalities in the retina with secondary effects on the formation of gel vitreous.

ADULT VITREOUS STRUCTURE

Vitreous Body

In an emmetropic adult human eye, the vitreous body is about 16.5 mm in axial length, with a depression anteriorly just behind the lens known as the patellar fossa (Figs. 10 and 12G). The hyaloideocapsular ligament of Weiger is the annular region of vitreolenticular attachment that is 1 to 2 mm in width and 8 to 9 mm in diameter. Erggelet's or Berger's space is at the center of the hyaloideocapsular ligament. Arising from this space and coursing posteriorly through the central vitreous is the canal of Cloquet (see Figs. 9B, 10, and 12G), the former site of the hyaloid artery in the embryonic vitreous. What was previously the lumen of this artery is an area devoid of collagen fibrils, surrounded by multifenestrated sheaths that were previously the basal laminae of the hyaloid artery wall. Posteriorly, Cloquet's canal opens into a funnel-shaped region anterior to the optic disc known as the area of martegiani. A remnant of incomplete atrophy of the hyaloid artery may persist in this location and is called Bergmeister's papilla.

Within the adult human vitreous, there are fine parallel fibers coursing in an anteroposterior direction that are continuous and do not branch (Fig. 12B and 12C). The fibers arise from the vitreous base (see Fig. 12H), where they insert anterior and posterior to the ora serrata. Although there are various concepts of how the peripheral anterior vitreous fibers connect with the retina and pars plana,^3,12 all espouse the precept that retinal tears result from vitreous traction on foci of strong adhesion at the vitreoretinal interface in these locations.¹⁴⁶ The fibers in the anterior vitreous base (Fig. 13) probably also have an important role in the pathogenesis of anterior proliferative vitreoretinopathy (PVR); not only in being the substrate on which fibronectin and laminin are deposited to facilitate cells to migrate, proliferate, and form PVR membranes but also in transmitting the traction induced by these membranes to the peripheral retina (producing retinal detachment; RD), to the ciliary body (causing detachment and hypotony), and even to the iris (resulting in the retraction commonly seen in PVR). Fibers near the vitreous cortex course posteriorly in a plane circumferential to the vitreous cortex, whereas central fibers undulate in a configuration that parallels the course of Cloquet's canal.

Ultrastructural studies have demonstrated that these fibers are composed of collagen fibrils organized into packed bundles of parallel fibrils (Fig. 14).⁸ The aggregation of collagen fibrils into visible fibers (Figs. 12 and 13) occurs when HA molecules no longer separate the collagen fibrils because of disease, such as the arthroophthalmopathies or diabetes, or aging. The areas adjacent to these large fibers have a low density of collagen and a relatively high concentration of HA and water. Comprised primarily of liquid vitreous, these areas scatter relatively little incident light.

Vitreoretinal Interface

The periphery of the vitreous body, known as the vitreous cortex, courses anteriorly and posteriorly from the vitreous base. The portion that courses forward and inward from the vitreous base is called the anterior vitreous cortex. The portion coursing posteriorly from the posterior border of the vitreous base is known as the posterior vitreous cortex. The posterior vitreous cortex is 100 to 110 μm thick and consists of densely packed type II collagen fibrils (Fig. 15) and other extracellular matrix components. Contrary to previous beliefs, there are no direct connections between the posterior vitreous cortex and the retina, yet the posterior vitreous cortex is adherent to the internal limiting lamina (ILL) of the retina, especially in youth (Fig. 16). The exact nature of the adhesion between the posterior vitreous cortex and the ILL is not known but most probably results from the action of the various extracellular matrix molecules found at this interface.^147,148

When the posterior vitreous is detached from the retina, a hole in the prepapillary vitreous cortex can often be seen (Fig. 17A). If peripapillary glial tissue is torn away during PVD and remains attached to the vitreous cortex surrounding the prepapillary hole, it is referred to as Vogt's or Weiss's ring. Vitreous can extrude through the prepapillary hole in the vitreous cortex (see Fig. 12A) but does so to a lesser extent than through the premacular vitreous cortex (see Fig. 12B and D). Various vitreomaculopathies can result from anteroposterior traction.^148,149 Other mechanisms, particularly tangential vitreomacular traction,^149,150 have been implicated in the pathogenesis of macular holes.

HYALOCYTES.

There are cells embedded within the posterior vitreous cortex (Fig. 18) known as hyalocytes. Situated 20 to 50 μm from the ILL of the retina, these mononuclear cells are widely spread apart in a single layer (Fig. 19). The highest density of hyalocytes is in the region of the vitreous base, followed by the posterior pole, with the lowest density at the equator. Derived from circulating monocytes and renewed every 6 months, hyalocytes are oval or spindle-shaped and are 10 to 15 μm in diameter. They contain a lobulated nucleus, a well-developed Golgi complex, smooth and rough endoplasmic reticula, many large periodic acid-Schiff-positive lysosomal granules, and phagosomes (Fig. 20). Balazs⁷² noted that hyalocytes are located in the region of highest HA concentration and suggested that these cells may be responsible for vitreous HA synthesis, which may also be true of vitreous collagen.

The phagocytic capacity of hyalocytes derives from the presence of pinocytic vesicles, phagosomes (Fig. 20), and cell membrane receptors that bind immunoglobulin G (IgG) and complement. It is intriguing to consider that hyalocytes are among the first cells to be exposed to any migratory or mitogenic stimuli released during various disease states. Because hyalocytes are essentially tissue macrophages, they may have a role similar to macrophages in other diseases, i.e., to recruit cells and elicit further escalation of the inflammatory response. Whereas in some tissues this may be desirable, in the vitreous this process results in various untoward events, depending on the clinical circumstances. In the case of pars planitis, hyalocytes in the vitreous base may be responsible for antigen processing and eliciting an immune response typical of tissue macrophages that leads to the observed snow banks of inflammatory cells in the peripheral anterior vitreous. Indeed, for these and other reasons, this condition may be more aptly named peripheral anterior vitritis. After failed surgery for rhegmatogenous RD, hyalocytes are likely the first cells to participate in the formation of the contractile membranes that in PVR detach the retina again. The particularly high density of hyalocytes in the vitreous base may be important in the pathogenesis of anterior PVR, wherein the prominence of vitreous fibers also contributes to the disease process by facilitating cell migration and transmitting traction to the peripheral retina, ciliary body, and iris. Thus, the role of these cells must be adequately considered in defining the pathophysiology of PVR, perhaps leading to a new avenue for early intervention.

The basal laminae about the vitreous body are composed of type IV collagen closely associated with glycoproteins.^148,152 At the pars plana, the basal lamina has a true lamina densa. Posterior to the ora serrata, the basal lamina is the ILL of the retina (Fig. 21). Within the ILL, the layer immediately adjacent to the Müller cell is a lamina rara, which is 0.03 to 0.06 μm thick. The lamina densa is thinnest at the fovea (0.01 to 0.02 μm) and disc (0.07 to 0.1 μm). It is thicker elsewhere in the posterior pole (0.5 to 3.2 μm) than at the equator or vitreous base. At the posterior pole, the anterior surface of the ILL (vitreous side) is smooth, whereas the posterior aspect is irregular, filling the spaces created by the irregular surface of the subjacent retinal glial cells (see Fig. 21A). In the periphery, both the anterior and posterior aspects of the ILL are smooth (see Fig. 21B). The significance of this topographic variation is not known.

At the rim of the optic disc, the retinal ILL ceases, although the basal lamina continues as the inner limiting membrane of Elschnig.¹⁵² This structure is 50 nm thick and is believed to be the basal lamina of the astroglia in the optic nerve head. At the most central portion of the optic disc, the membrane thins to 20 nm, follows the irregularities of the underlying cells of the optic nerve head, and is composed only of GAGs and no collagen.¹⁵² This structure is known as the central meniscus of Kuhnt. The thinness and chemical composition of the central meniscus of Kuhnt and the membrane of Elschnig may not be an adequate barrier to cell migration and proliferation. Consequently, this may explain the frequency with which abnormal cell proliferation arises from or near the optic disc in proliferative diabetic vitreoretinopathy and premacular membranes.

The posterior vitreous cortex is most firmly attached at the vitreous base, the disc and macula, and over retinal blood vessels. The posterior aspect (retinal side) of the posterior vitreous cortex is increasingly thick, in direct proportion to the distance from the ora serrata. Attachment plaques between the Müller cells and the ILL have been described in the basal and equatorial regions of the fundus but not in the posterior pole, except the fovea.^12,153 It has been hypothesized that these develop in response to vitreous traction on the retina. It has also been proposed that the thick ILL in the posterior pole dampens the effects of vitreous traction, except at the fovea where the ILL is thin. The thinness of the ILL and the purported presence of attachment plaques at the perifoveal central macula could explain the predisposition of this region to changes induced by traction, such as in macular holes.

There is an unusual vitreoretinal interface overlying retinal blood vessels. Physiologically, this may provide a shock-absorbing function, damping arterial pulsations. Pathologically, however, this structural arrangement may also explain the hemorrhagic and proliferative events associated with vitreous traction on retinal blood vessels during PVD and proliferative diabetic vitreoretinopathy.

RHEOLOGIC CHANGES

Rheology is a term that refers to the gel–liquid state of the vitreous. After the age of 45 to 50 years, there is a significant decrease in the gel volume and an increase in the liquid volume of human vitreous. Postmortem studies of dissected vitreous qualitatively confirmed these findings and determined that liquefaction begins in the central vitreous.^5,12 This finding supports the observation that the central vitreous is where fibers first appear (Fig. 12) and is consistent with the concept that breakdown of the normal collagen-HA association results in the simultaneous formation of liquid vitreous and aggregation of collagen fibrils into bundles of parallel fibrils seen as large fibers.^7,8,12 In the posterior vitreous, pockets of liquid vitreous are formed, recognized clinically as lacunae (Fig. 22). When a single large pocket forms, the terms bursa or precortical pocket are used.^6,9 As previously mentioned, the large lacuna is a manifestation of age-related liquefaction, or synchisis, and does not represent an anatomic entity.^10,11 Flood and Balazs¹⁵⁴ found evidence of liquid vitreous after the age of 4 years and observed that by the time the human eye reaches adult size (age 14 to 18 years), 20% of the total vitreous volume consists of liquid vitreous (Fig. 23). In these postmortem studies of fresh unfixed human eyes, it was noted that after the age of 40 years, there is a steady increase in liquid vitreous. This occurs simultaneously with a decrease in gel volume. By the ages of 80 to 90 years, more than half of the vitreous is liquid.

Vitreous liquefaction does not occur in most mammals¹⁵⁴ and only a few species develop fibers.¹¹⁸ Studies¹⁵⁴ have demonstrated that in rhesus monkeys, however, an age-related process of liquefaction occurs that is similar to that in humans. In the owl monkey, there is a gel-to-liquid transformation that occurs by the age of 2 years (human age, 8 to 10 years). In the baby owl monkey, the vitreous has a gel structure and contains a network of collagen fibrils, whereas in the adult there is disappearance of collagen and a simultaneous transformation from a gel state to a viscous fluid that contains mostly high molecular weight HA.¹⁵⁴

The mechanism of vitreous liquefaction is poorly understood. Gel vitreous can be liquefied in vitro by removing collagen via filtration,¹⁵⁵ centrifugation,¹⁵⁶ or by enzymatic destruction of the collagen network.¹⁵⁷ The importance of collagen in the maintenance of the gel state of vitreous can be deduced from the biochemical findings in the owl monkey, wherein the gel state is lost simultaneous to replacement of vitreous collagen by HA.¹¹² Studies using nuclear magnetic resonance (NMR) spectroscopy in experiments performed with bovine vitreous found that collagenase induced a decrease in macroscopic viscosity and a shortening of longitudinal and transverse relaxation times.¹⁵⁸ The decrease in viscosity was proportional to the shortening of relaxation times for collagenase-treated eyes—more than for hyaluronidase-treated eyes. This suggests that the transition from gel to liquid vitreous is in some way related to disappearance or displacement of collagen from the collagen-HA double network, resulting in a decrease in viscosity and liquefaction, as detected by NMR spectroscopy. These findings are consistent with the hypotheses invoking collagen dissociation from HA with aggregation of collagen fibrils into bundles and pooling of HA and water into lacunae as a way to explain the observed structural changes noted during aging.

Primary changes in the conformation of HA molecules with secondary cross-linking of collagen molecules and aggregation of fibrils into bundles is another possible mechanism of endogenous vitreous liquefaction. Chakrabarti and Park⁵³ claimed that the interaction between collagen and HA depends on the conformation of each macromolecule and that a change in the conformation of HA molecules may result in alterations in collagen and pooling of HA, drawing water along and resulting in vitreous liquefaction. Armand and Chakrabarti¹⁵⁹ detected differences in the structure of the HA molecules present in gel vitreous when compared with liquid vitreous, suggesting that conformational changes occurred during aging, resulting in liquefaction. Andley and Chapman¹⁶⁰ have demonstrated that singlet oxygen can induce conformational changes in the tertiary structure of HA molecules. Ueno and associates⁸² have suggested that free radicals generated by metabolic and photosensitized light-tissue interactions may alter HA or collagen structure and trigger a dissociation of collagen and HA molecules, ultimately leading to liquefaction. This is plausible because the cumulative effects of a lifetime of daily exposure to light could indeed influence the structures and interaction of vitreous collagen and HA by the proposed free radical mechanism. In recognition of the important role of HA in maintaining vitreous structure, investigators attempting to induce liquefaction have used exogenous hyaluronidase injection in a rabbit model.¹⁶¹ However in humans, studies attempting to clear vitreous hemorrhage have had disappointing results with exogenous hyaluronidase injection because a phase III FDA trial recently failed to show efficacy.

Vitreous liquefaction may also be the result of changes in the minor GAGs and chondroitin sulfate profile of vitreous.¹⁶² In these studies on rabbit vitreous, liquefaction was associated with the disappearance of lower-charged chondroitin sulfate and the appearance of a higher-charged chondroitin sulfate. Such observations have led to attempts to alter vitreous rheology and structure by injecting chondroitinase ABC to purportedly break down chondroitin sulfate and induce liquefaction and disinsertion of the vitreous body.¹⁶³ These findings have led to the study of chondroitinase as an adjunct to vitreous surgery, intended to facilitate removal of vitreous (see below).

BIOCHEMICAL CHANGES

Biochemical studies by Flood and Balazs¹⁵⁴ support the rheologic observations described above. Total collagen content in vitreous does not change after the ages of 20 to 30 years (Fig. 7). In a large series of normal human eyes obtained at autopsy, however, the collagen content in the gel vitreous at the ages of 70 to 90 years (approximately 0.1 mg) was significantly greater than at the ages of 15 to 20 years (approximately 0.05 mg; p > 0.05). Because the total collagen concentration does not change during this time, these findings are most likely the result of the decrease in the volume of gel vitreous that occurs with aging and a consequent increase in the concentration of the collagen in the remaining gel. Without the previously intercalated HA molecules, the collagen fibrils in this gel aggregate and become packed into bundles of parallel fibrils (Figs. 12 and 14).^7,8 Because HA is hydrophillic, the HA molecules that are no longer enmeshed in the collagen network form liquid vitreous. This concept is supported by the finding that vitreous HA concentration increases until approximately the age of 20, when adult levels are attained (Fig. 8). Thereafter, from the ages of 20 to 70 years, there are no changes in the HA concentrations of either the liquid or gel compartments. This means that there is an increase in the HA content of liquid vitreous and a concomitant decrease in the HA content of gel vitreous because the amount of liquid vitreous increases and the amount of gel vitreous decreases with age (Fig. 23).

STRUCTURAL CHANGES

As a result of the rheologic and biochemical alterations, there are significant structural changes in the aging vitreous. These consist of a transition from a clear vitreous in youth (Figs. 9 and 11), which is the result of a homogeneous distribution of collagen and HA, to a fibrous structure in the adult (Fig. 12). This is the result of collagen fibril aggregation that occurs when HA is dissociated from the macromolecular network, which maintains transparency and the gel state (Fig. 1). In old age, the advanced liquefaction, thickening and tortuosity of vitreous fibers and collapse of vitreous body (Fig. 22) result in PVD (Fig. 17). For this process to occur innocuously, there must be a weakening of vitreoretinal adhesion, allowing liquid vitreous to dissect a plane between the posterior vitreous cortex and the ILL of the retina.

VITREORETINAL INTERFACE CHANGES

As occurs in basal laminae throughout the body, there is age-related thickening of the basal laminae that surround the vitreous body.¹⁶⁴ This is believed to be the result of synthesis by retinal Müller cells, perhaps as a result of traction induced on the inner retina over a period of many years by the attached posterior vitreous cortex.^165,166 Such thickening of the ILL may ultimately contribute to weakened vitreoretinal adherence, which is a necessary, but not sufficient, element in the pathogenesis of PVD.

Teng and Chi¹⁶⁷ found that the width (in the radial dimension) of the vitreous base posterior to the ora serrata increased with age to nearly 3.0 mm, bringing the posterior border of the vitreous base closer to the equator. This widening of the vitreous base was found to be most prominent in the temporal portion of the globe. The phenomenon of posterior migration of the posterior border of the vitreous base has recently been confirmed and an explanation for increased vitreoretinal adhesion in this location has been offered.¹⁶⁸ It appears that there is intraretinal synthesis of collagen fibrils that penetrate the internal limiting lamina of the retina and splice with vitreous collagen fibrils. Such posterior migration of the vitreous base may have an important role in the pathogenesis of peripheral retinal breaks and rhegmatogenous RD. Gartner¹⁶⁹ found lateral aggregation of the collagen fibrils in the vitreous base of older individuals, similar to changes within the central vitreous. These changes at the vitreous base may lead to increased traction on the peripheral retina, which, in the setting of a vitreous base that is inserted more posteriorly in the retina, may lead to the development of retinal tears and detachment after PVD.

POSTERIOR VITREOUS DETACHMENT

Defined as a separation of the posterior vitreous cortex from the ILL of the retina, PVD is the most common event to occur in the human vitreous. PVD can be localized, partial, or total (up to the posterior border of the vitreous base). Autopsy studies found that the incidence of PVD in the eighth decade is 63%.¹⁷⁰ PVD is more common in women and in the presence of myopia, occurring 10 years earlier than in emmetropia and hyperopia.¹⁷¹ Cataract extraction in myopic patients introduces additional effects, causing PVD in all but 1 of 103 myopic (greater than –6 diopters) eyes in a 1975 study of intracapsular surgery.¹⁷²

PVD occurs when there is weakening of the vitreous cortex-ILL adhesion in conjunction with liquefaction (synchisis) of the vitreous body. Dissolution of the vitreous cortex-ILL adhesion at the posterior pole allows liquid vitreous to enter the retrocortical space by the prepapillary hole and perhaps even the premacular vitreous cortex. With ocular saccades and rotational eye movements, liquid vitreous acts as a wedge to dissect a plane between the vitreous cortex and the ILL, leading to true PVD. It is possible that in certain circumstances (e.g., myopia and the inherited disorders of type II collagen metabolism found in various arthroophthalmopathies), there is either an increased amount of liquid vitreous or an alteration in the biochemical constituents of the liquid vitreous (or both) that facilitates vitreoretinal separation.^172,173The volume displacement of liquid vitreous from the central vitreous to the preretinal space causes the observed collapse of the vitreous body (syneresis).

Although PVD may induce glare caused by light scattering by the dense collagen fibril network in the posterior vitreous cortex, the most common complaint of patients with PVD is floaters. This usually results from entoptic phenomena caused by condensed vitreous fibers, glial tissue of epipapillary origin that adheres to the posterior vitreous cortex, or intravitreal blood. Floaters move with vitreous displacement during ocular saccades and scatter incident light, casting a shadow on the retina that is perceived as a gray hairlike or flylike structure. When the ocular saccade stops, the detached posterior vitreous cortex continues to move slightly, giving rise to the subjective experience described as a floater. Wise¹⁷⁴ noted that Moore's light flashes, first described in 1935, occurred in 50% of cases at the time of PVD and were usually vertical in orientation and temporal in location. Generally thought to result from vitreoretinal traction, these photopsia are considered by most to signify a higher risk of retinal tears. Voerhoeff,¹⁷⁵ however, suggested that the light flashes actually result from the detached posterior vitreous cortex impacting the retina during ocular saccades. This theory is not inconsistent with an increased risk of retinal tears in an individual with PVD complaining of photopsia, although the tears might be on the same side of the photopsia as opposed to the opposite side, if traction is the cause of the photopsia.

VITREOUS LIQUEFACTION (NONAGING)

Disorders of Collagen Metabolism

The role of collagen in maintaining a gel vitreous structure has been described above. Thus, it follows that inborn errors of type II collagen metabolism have profound effects on vitreous structure. As noted, that both vitreous and articular cartilage are rich in type II collagen explains why metabolic abnormalities in type II collagen metabolism affect both of these tissues simultaneously. Maumenee¹⁷³ has characterized several such disorders with autosomal dominant inheritance and identified them as single-gene diseases with dysplastic connective tissues resulting in joint laxity and various other skeletal abnormalities. Marfan's, Ehlers-Danlos, and Stickler are the best known of these syndromes. Advanced vitreous liquefaction at a young age is typical in these patients. Because there is no concomitant dehiscence at the vitreoretinal interface, there is a high incidence of retinal detachment caused by large posterior tears. A study¹⁷⁶ of Marfan's syndrome in the Czech Republic found that of 18 eyes with retinal detachment, 5 of 18 (28%) had tears between 80 and 120 degrees in size, 11 of 18 (61%) had tears at or posterior to the equator, and 12 of 18 (68 %) had detachments of three quadrants or more. This last-mentioned probably results form the extensive amounts of liquefied vitreous available to enter the subretinal space.

Diabetic Vitreopathy

Christiansson¹⁷⁷ showed that alloxan-induced experimental diabetes in rabbits resulted in an increase in glucosamine content and viscosity of vitreous and a decrease in vitreous volume. More recent studies¹⁷⁸ showed a slight increase in the tonicity of human diabetic vitreous (324 ± 23 mOsm versus 316 ± 21 mOsm in controls). Lundquist and Osterlin¹⁷⁹ have shown that in humans with diabetes, there is an increase in vitreous glucose levels. These elevated levels of glucose were found by Sebag and colleagues¹⁸⁰ to be associated with increased nonenzymatic glycation products in human vitreous and elevated levels of the enzyme-mediated cross-link dihydroxylysinonorleucine. The advanced glycation end products (AGEs) have been further characterized by Fourier transform Raman spectroscopy¹⁸¹ and are believed to be the cause of structural alterations⁹³ that constitute diabetic vitreopathy⁵⁷ (Fig. 24). Subsequent studies¹⁸² confirmed the original biochemical findings and also showed that the glycation of vitreous proteins (primarily collagen) can be inhibited by aminoguanidines.

There may be considerable effects of diabetes on vitreous GAGs as well, not only in terms of nonenzymatic glycation effects on the protein core of the proteoglycans, but also on the GAGs themselves. Studies have shown that the presence of AGEs, promotes a decrease in the molecular weight of HA when exposed to light.¹⁸³ The investigators proposed that the photosensitizer property of AGEs may be associated with accelerated depolymerization of HA in diabetic patients, contributing to diabetic vitreopathy.⁵⁷

Shifts in systemic osmolarity and hydration that occur as a result of poor glycemic control can alter the ionic milieu in and around the vitreous body, extending or contracting vitreous HA, and in turn the vitreous body. This can transmit traction to structures attached to the posterior vitreous cortex, such as new blood vessels that have grown out of the optic disc or retina (Fig. 25).¹⁸⁴ These events may influence the course of diabetic retinopathy by stimulating further proliferation of neovascular fronds and perhaps even by inducing rupture of these new vessels, resulting in vitreous hemorrhage. Tasman¹⁸⁵ found that in 53 cases of vitreous hemorrhage caused by proliferative diabetic retinopathy, 62.3% of bleeding episodes occurred between midnight and 6 A.M., whereas the remaining parts of the day had only an 11% to 13% incidence. Although Tasman speculated that this may be the result of nocturnal hypoglycemia, sleep apnea is perhaps a more plausible mechanism.

ANOMALOUS POSTERIOR VITREOUS DETACHMENT

Anomalous posterior vitreous detachment¹³ (APVD) occurs when the extent of vitreous liquefaction exceeds the degree of weakening of vitreoretinal adherence and traction is exerted at this interface. There are various causes for an imbalance between the degree of gel liquefaction and weakening of vitreoretinal adhesion. As described above, inborn errors of collagen metabolism, such as those present in Marfan's, Ehlers-Danlos, and Stickler syndromes¹⁸⁶ result in extreme gel liquefaction at an early age when there is persistent vitreoretinal adherence. The result is a high incidence of large retinal tears and detachments. Systemic conditions such as diabetes induce biochemical¹⁸⁰ and structural⁹³ alterations in vitreous. These changes, referred to as diabetic vitreopathy,⁵⁷ are important in the pathobiology of proliferative diabetic vitreoretinopathy, and perhaps some cases of macular edema as well. Diabetic vitreopathy may one day be detected in vivo using noninvasive optical instrumentation.^181,187,188 There are also changes associated with myopia, known as myopic vitreopathy, where there is excess vitreous liquefaction for the degree of vitreoretinal adhesion, resulting in anomalous PVD and undue traction at the vitreoretinal interface.¹⁸⁹

Regardless of the underlying cause, abnormal traction at the vitreoretinal interface can have deleterious effects upon retina as well as vitreous (Table 1).

TABLE 1. Anomalous Posterior Vitreous Detachment

Retinal Effects

Effects upon the retina vary, depending on the site affected. These include hemorrhage, retinal tears and detachment, and vitreomacular traction syndromes, including macular holes and some cases of diabetic macular edema. Proliferative diabetic retinopathy (PDR) can be aggravated by anomalous PVD. Lindner¹⁷¹ found that vitreous hemorrhage occurred in 13% to 19% of patients with PVD. Because vitreous hemorrhage results from considerable vitreoretinal traction, this finding in a patient with PVD is generally considered to be an important risk factor for the presence of a retinal tear and detachment. One 15-year study in Belgium¹⁹⁰ found that in 126 cases of nondiabetic, nontraumatic vitreous hemorrhage that did not clear for 6 months, 25% were found to have retinal tears, and 8% had retinal detachments. Another study¹⁹¹ found that in 36 eyes with fundus-obscuring vitreous hemorrhage, 24 of 36 eyes (67%) were found to have at least one retinal break, with 88% of breaks located in the superior retina. Eleven eyes (31%) had more than one retinal break. Fourteen of 36 eyes (39%) had a rhegmatogenous retinal detachment.

Retinal tears not involving blood vessels result from traction on other foci of usual vitreoretinal adhesion, such as the posterior border of the vitreous base. Abnormal foci of firm vitreoretinal adhesion, such as lattice degeneration and rosettes, are also frequently associated with retinal tears after PVD. Indeed, Byer¹⁹² has claimed that as many as 25% of the general population have some form of abnormal focal vitreoretinal adhesion, placing them at considerable risk from anomalous PVD.

Vitreous Effects

Effects upon vitreous primarily involve posterior vitreoschisis¹⁹³ (Fig. 26). This condition results from splitting of the posterior vitreous cortex, with forward displacement of the anterior portion of the posterior vitreous cortex leaving part, or all, of the posterior layer of the split vitreous cortex still attached to the retina.¹⁹⁴ Vitreoschisis has been detected in cases of proliferative diabetic vitreoretinopathy¹⁹⁵ and likely has a role in the pathophysiology and sequelae of this condition. Premacular membranes with macular pucker and cases of macular holes may also result from persistent attachment of part or all of the posterior vitreous cortex to the macula while the remainder of the vitreous detaches forward. In the former case, tractional forces are centripetal (inward toward the fovea) causing macular pucker. In the latter condition tangential traction occurs in a centrifugal (outward from the fovea) direction, causing a macular hole.

VITREOUS OPACIFICATION (NON-HEMORRHAGIC)

Asteroid Hyalosis

This benign condition is characterized by small white or yellowish white spherical or disc-shaped opacities throughout the vitreous (Fig. 27). Asteroid bodies are so intimately enmeshed within vitreous gel that they move in a manner typical of vitreous displacement during ocular saccades. This observation led Rodman and co-workers¹⁹⁶ to suggest that there was a relation to vitreous fibril degeneration. PVD, however, occurs less frequently in asteroid hyalosis than in age-matched controls, so this observation does not support age-related degeneration as a cause.¹⁹⁷ The Beaver Dam eye study of 4,747 subjects found that asteroid hyalosis was present in 1.2%.¹⁹⁸ In subjects in whom it was present, asteroid hyalosis was bilateral in 9%. The prevalence increased significantly (p < 0.001) with age from 0.2% in subjects 43 to 54 years to 2.9% in subjects 75 to 86 years. There was no association with diabetes. This was confirmed in the Blue Mountains Eye Study of 3,654 subjects in Australia¹⁹⁹ where a prevalence of 1% was identified, bilateral occurrence was found in 8.3%, and no association with diabetes could be identified. Asteroid hyalosis can be associated with certain pigmentary retinal degenerations.²⁰⁰

Histologic studies detected a crystalline appearance to asteroid bodies that stained positive to fat and acid mucopolysaccharide stains and was unaffected by pretreatment with hyaluronidase.²⁰¹ Ultrastructural studies found intertwined ribbons of multilaminar membranes with a 6-nm periodicity that was interpreted as characteristic of complex lipids, especially phospholipids, lying in a homogeneous background matrix.²⁰² In these investigations, energy-dispersing radiographic analysis showed calcium and phosphorus to be the main elements in asteroid bodies. Electron diffraction structural analysis demonstrated calcium hydroxyapatite and possibly other forms of calcium phosphate crystals. Yu and Blumenthal²⁰³ proposed that asteroid hyalosis results from aging collagen, with sulfated GAGs acting as chelators in the binding of calcium to free polar groups on vitreous collagen fibrils. Experimental studies in the rabbit, however, suggested that asteroid formation is preceded by depolymerization of HA.²⁰⁴

More recent studies²⁰⁵ used electron energy loss spectroscopy and energy-filtered transmission electron microscopy for the elemental analysis of asteroid bodies. The ultrastructural localization of glycosaminoglycans was investigated using lectin and antibody conjugates in conjunction with transmission electron microscopy and epifluorescence microscopy. Anionic sites of glycosaminoglycans were detected with 15 nm of cationic colloidal gold at low pH, applied as a postembedding technique. Ultrastructural details of asteroid bodies were documented using fast Fourier transform analysis of zero-loss filtered images. The results suggested that it was more specifically the GAG side chains of vitreous proteoglycans that were important in regulating this biomineralization process. The mineralization was confirmed by chemical analysis of samples removed at surgery performed with an energy-dispersive x-ray microanalyzer studies²⁰⁶. The observed changes resembled stones in the living body (lithiasis), leading the investigators to conclude that asteroid hyalosis is a condition of lithiasis in the vitreous body.

Although vision is rarely affected by asteroid hyalosis, the presence of asteroid bodies in vitreous interferes with ultrasonographic measurements of axial length.²⁰⁷ The artefactual lowering of axial length measurements that results from this effect may lead to errors in calculating the power of intraocular lenses.

Amyloidosis

Systemic amyloidosis can result in the deposition of opacities in the vitreous of one or both eyes. Bilateral involvement can be an early manifestation of the dominant form of familial amyloidosis. The opacities initially appear in the posterior vitreous cortex anterior to retinal blood vessels and later appear in the anterior vitreous. Amyloid opacities are at first granular, with wispy fringes that later take on a glass wool appearance. When the opacities form strands, they appear to attach to the retina and the posterior aspect of the lens by thick footplates.²⁰⁸ After PVD, the posterior vitreous cortex appears to have linear opacities that are thick and follow the course of the retinal vessels. The opacities seem to aggregate by seeding on vitreous fibrils and along the posterior vitreous cortex.²⁰² In a recent study²⁰⁹ of patients with familial amyloidotic polyneuropathy, the prevalence of vitreous opacities was much higher in patients with one genetic subtype (100% in ATTR Tyr114Cys) than another (24% in ATTR Val30 Met). The mean age at the onset of vitreous opacities was also significantly lower in the patients with the former subtype than the latter (p < 0.005).

Opacified vitreous caused by amyloidosis can be safely removed surgically. Specimens that have been studied histopathologically contained starlike structures with dense fibrillar centers. Amyloid fibrils are 5 to 10 nm in diameter and are distinguishable from vitreous fibrils by stains for amyloid and because the vitreous fibrils are straight and long. Electron microscopic studies have confirmed the presence of amyloid, whereas immunocytochemical studies have identified the major amyloid constituent as a protein resembling prealbumin.²¹⁰ Streeten²⁰¹ has proposed that hyalocytes may perform the role of macrophage processing of the amyloid protein before its polymerization, which may explain why these opacities initially appear at the posterior vitreous cortex where hyalocytes reside.

CHANGES AFTER CATARACT SURGERY

The incidence of retinal detachment (RD) in the general population ranges between 0.005% and 0.01%.²¹¹ After intracapsular cataract extraction, the incidence was 1.74%, considerably higher than the incidence of 0.62% after extracapsular surgery. However, neodymium:yttrium-aluminum-garnet (Nd:YAG) capsulotomy increases the risk of RD, almost fivefold in one study.²¹² The molecular and structural effects of cataract surgery and posterior capsulotomy on vitreous provide insight into understanding the changes that contribute to the pathogenesis of RD in these patients.

Effects of Cataract Surgery on Vitreous

After intracapsular cataract extraction, there is marked reduction in vitreous HA concentration compared to the fellow phakic eyes of the same individuals.²¹³ This probably results from HA diffusion out of the vitreous body into the anterior segment.²¹⁴ Based on these findings, Osterlin^213,214 proposed that aphakic eyes are more predisposed to develop retinal tears and detachments because the decrease in vitreous HA concentration causes a decrease in vitreous viscosity, allowing more vitreous movement with ocular saccades. The increased traction on the vitreous base produces retinal tears, which result in retinal detachments that rapidly enlarge to extensive detachments. This hypothesis is supported by the observation that in contrast to phakic patients with RD, patients with RD after cataract surgery usually have small retinal tears at the posterior border of the vitreous base.²¹⁵ Despite these tears being small, the decreased vitreous viscosity resulting from decreased HA concentration enables easier and more rapid hydraulic access of liquid vitreous to the subretinal space by these small retinal tears, producing RD more often, more rapidly, and more extensively. These observations apply only to cases of cataract surgery by intracapsular techniques, in which there was about a 90% reduction in HA concentration.²¹³ After extracapsular cataract extraction with an intact posterior capsule, there is no appreciable change in vitreous HA concentration. This was confirmed in a postmortem study of human eyes that compared HA concentrations in a pseudophakic eye with the fellow phakic eye; there was significant reduction in HA concentration after intracapsular extraction and no change after extracapsular surgery.²¹⁶ These findings likely explain the decreased incidence of retinal tears and detachments after cataract surgery by extracapsular techniques –in one study by as much as 50%.²¹⁷

Effects of Posterior Capsulotomy on Vitreous

Creating an opening in the posterior capsule by Nd:YAG laser therapy induces changes in the vitreous similar to those observed after intracapsular cataract extraction. The molecular changes are likely due to escape of various vitreous molecules via the capsulotomy site. Experimental studies using tracers injected into the vitreous body found a tenfold increase in the forward movement of 20,000 dalton dextran into the anterior chamber after posterior capsulotomy.²¹⁸ Fluorophotometry confirmed these findings in humans undergoing Nd:YAG laser capsulotomy and found that intraocular pressure elevation occurred more frequently when the posterior capsule or anterior vitreous cortex barrier complex was altered,²¹⁹ suggesting that vitreous macromolecules increase aqueous viscosity, decrease aqueous outflow facility, and elevate intraocular pressure. The vitreous component most likely responsible for the increase in intraocular pressure after discission of the posterior capsule or anterior vitreous cortex complex is HA, which lowers vitreous HA concentration as it leaves.²²⁰ Because of the loss of vitreous HA, there is increased vitreous collagen fiber cross-linking into bundles of parallel collagen fibrils (Figs. 11 and 13), which destabilizes the supramolecular organization of vitreous liquefying vitreous and contributing to the development of PVD. Indeed, postmortem studies of human eyes status post cataract surgery found that PVD was twice as common in eyes with open posterior capsules.²²¹ Considering the importance of PVD in the pathogenesis of retinal tears and detachments, it is understandable that in one clinical, study eyes undergoing primary capsulotomy had a threefold higher incidence of postoperative RD than controls.²²² In another study of 57,103 patients who underwent extracapsular cataract extraction, proportional-hazards modeling showed an almost fourfold increase in the risk of retinal break or detachment among those who subsequently underwent posterior capsulotomy by Nd:YAG laser therapy.²²³ Compared to patients older than 85 years, those 75 to 84 years old had a threefold higher risk of RD, whereas those 65 to 74 years old had a sevenfold higher risk. This finding was confirmed in a subsequent study²²⁴ where it was also determined that the patients who developed RD after cataract surgery were about 20 years younger than those who did not develop RD (p < 0.001). This is probably because at the time of surgery older patients are more likely to have already experienced PVD, decreasing the chances that they will develop retinal detachment after cataract surgery and posterior capsulotomy. Younger individuals, however, are at greater risk because they have not yet had a PVD, placing them at greater risk of developing anomalous PVD and retinal detachment. Interestingly, this study also found that diabetes was associated with a twofold increase in the risk of retinal tears or detachments (p > 0.05), possibly the consequence of diabetic vitreopathy.⁵⁷ Indeed, this nested case-controlled study²²⁴ of 291 cases of RD and 870 matched controls used conditional logistic regression models and found that in addition to Nd:YAG capsulotomy, other risk factors for RD included lattice degeneration, ocular trauma after cataract surgery, prior history of RD, axial length, and refractive error.

Thus, it appears that cataract surgery induces significant molecular and structural changes within vitreous that have an important role in the development of retinal tears and detachment postoperatively. Furthermore, there is a significantly higher risk of retinal tears and detachments after cataract surgery by techniques that do not maintain the integrity of the posterior capsule, especially in younger individuals and patients with diabetes. Thus, the choice of surgical technique for cataract extraction and the decision to perform the seemingly benign procedure of posterior capsulotomy by Nd:YAG laser therapy must be considered with increased forethought and caution.

CHANGES ASSOCIATED WITH MYOPIA

Although one of every four people in the United States is myopic, the prevalence of myopia is probably even higher in other parts of the world. A spherical equivalent refractive error of minus diopters is the most common method to define myopia. By this criterion, it has been estimated that there is a 4% prevalence of high myopia (greater than −6 diopters) in the general population.²²⁵ This makes myopia one of the leading threats to vision because its effects on vitreous and the retina contribute considerably to the pathogenesis of RD. In one study, the risk of so-called idiopathic rhegmatogenous RD was found to be increased four-fold in individuals with a spherical equivalent refractive error of −1 to −3 diopters.²²⁶ For individuals with more than −3 diopters of myopia, the risk of RD was ten times greater than for nonmyopic controls. These investigators concluded that nearly 55% of nontraumatic RDs in eyes without previous ocular surgery are because of myopia. As astounding as these figures seem, they may be underestimated because, as noted by Percival,²²⁷ it is probably more accurate to define myopia based on axial length, particularly as it relates to the posterior segment complications of this condition. In his study, Percival found that using the cutoff for high myopia of −6 diopters yielded a 1.6% incidence of RD after cataract surgery. With the definition of high myopia as an axial length of more than 26.5 mm, however, the incidence of RD was found to be 4.1%. To understand the origin of these phenomena, it is necessary to consider myopia in terms of its effects on vitreous and retinal structure and the role of these changes in the pathogenesis of RD. It is also important to consider the combined effects of myopia and cataract surgery as contributing factors in the pathogenesis of postoperative RD, especially concerning the performance of clear lens extraction for high myopia.

Myopic Vitreopathy

The vitreous body in myopia is liquefied and contains filaments with localized nodules.²²⁸ Biochemical studies in myopic human eyes found decreased collagen content and concentration in the central vitreous.²²⁹ Hexosamine concentration, an index of HA, was decreased not only in the central vitreous but in the posterior vitreous cortex. Studies in experimental models of myopia also found a decrease in vitreous protein concentration.²³⁰ It was subsequently determined that the decrease in protein concentration in the liquid vitreous was concurrent with an increase in the protein concentration of the gel vitreous.²³¹ These studies also determined that although overall vitreous volume increased in experimental myopia, the volume of gel vitreous remained the same and there was a marked increase in liquid vitreous volume. Comparing the protein content in the liquid and gel vitreous of myopic eyes revealed that although the protein profiles of the gel and liquid vitreous were similar on the day of hatching, they differed by day 14 after hatching. Based on these findings, it would seem that the accumulation of liquid vitreous in myopic eyes is not a degenerative phenomenon but the result of active synthesis of liquid vitreous. This is different from the mechanism of vitreous liquefaction seen in aging, wherein the increase in liquid vitreous volume occurs in synchrony with a decrease in gel volume (see Fig. 23). It is also possible that this abundant synthesis of liquid vitreous may be the source of an increase in the pressure gradient differential between vitreous and the suprachoroidal space that contributes to elongation of the eye and myopia.^228,232

In a retrospective histopathologic study of 308 eyes with pathologic myopia, nonspecific degenerative changes of the vitreous were detected in 35.1%.²³² Although it is likely that these findings do not result from age-related degeneration of gel vitreous into liquid (synchisis) but from abnormal liquid vitreous formation, the structural manifestations of these two different processes may be indistinguishable by current evaluation techniques. Stirpe and Heimann²³⁴ found that in 87 of 496 (17.5%) highly myopic eyes, there were prominent posterior vitreous lacunae. These eyes had more prominent posterior staphyloma and a thin posterior vitreous cortex that was firmly adherent to the ILL of the retina. Posterior retinal breaks, including macular holes, were present in 56% of these cases. The formation of liquid vitreous in myopia markedly destabilizes vitreous and threatens the retina because this process occurs relatively early in life and is not concurrent with dehiscence at the vitreoretinal interface. It is known that in youth there is strong adhesion of the posterior vitreous cortex to the ILL of the retina (see Fig. 16).¹⁴⁷ Consequently, the findings that the degree of vitreous liquefaction and the incidence of PVD are greater in high myopia (more than −6 diopters) and the observation that PVD occurs 5 to 10 years earlier in myopic eyes than in emmetropia portend a poor prognosis for retinal tears and RD.^235,236 Furthermore, there are often distinct peripheral retinal lesions in myopia that make PVD a particularly ominous event.

Myopia Effects on the Retina

Physical examination of 165 eyes in patients with pathologic myopia detected lattice degeneration in 9.7% and retinal holes and tears in 3.6% of individuals.²³⁷ Histologic evaluation of 308 eyes with pathologic myopia revealed peripheral retinal degeneration in 30.6%, cobblestone degeneration in 14.3%, and somewhat surprisingly, lattice degeneration in only 4.9%.²³³ A variant of this type of degeneration was present in an additional 11.4%, however, bringing the true prevalence closer to 16.5%, which was the finding in a clinical study of 436 eyes with myopia of more than −6 diopters.²³⁸ Interestingly, this clinical study found that the highest prevalence (63/154 eyes; 40.9%) of lattice degeneration was in eyes with −6 to −8.7 diopters (axial length of 26 to 26.9 mm), and that the lowest prevalence (5 of 71; 7%) was in eyes with more than −24 diopters (axial length more than 32 mm). The investigators proposed no explanation for this finding but suggested that this may explain why RD after cataract surgery is seen more commonly in patients with moderate myopia than in those with severe myopia.

As previously mentioned, retinal detachment frequently occurs in patients with myopia.¹⁸⁵ A population-based case-controlled study²²⁴ comparing 291 cases of RD to 870 matched controls found that the odds ratio of RD increased by 0.92 for each diopter of myopia and by 1.21 for each millimeter of axial elongation, once again uderscoring the greater accuracy of defining myopia based on axial length, at least as it concerns the development of RD.²²⁷ Another study²³⁸ retrospectively compared 1166 eyes with RD to 11,671 eyes without RD. Myopia was present in 82.2% of RD patients, compared to 34.4% of controls. These phenomena are likely because of the myopic vitreopathy, younger age at onset of PVD in myopic individuals, relatively high incidence of peripheral retinal pathology in myopia, and the abundance of liquid vitreous available to access the subretinal space and detach the retina. Various other factors, especially cataract surgery, may further increase the risk of RD.

Cataract Surgery in Myopia

The Blue Mountains Eye Study found evidence of an association between myopia and incident cataract and cataract surgery, after adjustment for multiple confounders and severity of nuclear opacity.²³⁹ It is now known that vitreous undergoes considerable molecular and structural changes after cataract surgery.²⁴⁰ These abnormalities add to the risks of RD already present as a result of myopia (see above). Jaffe and associates²⁴¹ noted that in high myopia, the rate of RD is nine times greater after intracapsular cataract extraction than after extracapsular surgery. Performing extracapsular cataract extraction in high myopia without implanting an intraocular lens doubles the incidence of postoperative RD.²⁴² The incidence of postoperative RD in this group also doubles after Nd:YAG laser capsulotomy.²⁴³ While one study in Spain found that the risk of RD following clear lens extraction with posterior chamber IOL implantation is only 2.1% in 190 eyes,²⁴⁴ another in France found the incidence to be 8.1%, in spite of the fact that some of these cases had undergone prophylactic peripheral retinal laser photocoagulation.²⁴⁵ A study²⁴⁶ in Italy found that laser prophylaxis may not be very effective because 26 of 41 eyes that developed RD after clear lens extraction had been previously treated with 360-degree peripheral retinal laser prophylaxis. These observations suggest that particular caution needs to be exercised in contemplating, planning, and executing lens surgery in myopic patients, particularly concerning clear lens extraction.²⁴⁷ The future may see new treatments to prevent post-operative RD in myopia based upon pharmacologic alteration of the molecular structure of vitreous.

Pharmacologic vitreolysis was first attempted more than a half century ago when hyaluronidase was used in 1949,²⁴⁸ followed by collagenase in 1973.²⁴⁹ Indeed, in 1983 the latter agent was proposed²⁵⁰ as a means by which to assist vitrectomy. Table 2 outlines the approaches that are currently being developed. As indicated in this table, the different pharmacologic agents can be broadly grouped as enzymatic and nonenzymatic. Within the enzymatic group there are substrate-specific agents and nonspecific agents.

TABLE 2. Pharmacologic Vitreolysis Agents

A clinically-oriented way to consider the various agents being developed for pharmacologic vitreolysis is presented in Table 3 and described below.

TABLE 3

VITREORETINAL SURGERY ADJUNCTS

These agents are being developed primarily as adjuncts to facilitate vitreous surgery as it is currently being performed. Early observations on the effects of blood upon vitreous laid the groundwork for approaches based upon extracting active agents from blood for pharmacologic vitreolysis. Plasmin is a nonspecific protease that has been isolated from the patient's own serum for use at surgery. It has been tested in rabbits²⁵¹ and several small series of patients with macular holes^252,253 and diabetic retinopathy²⁵⁴ undergoing vitrectomy. To date, this agent has only been advocated as an adjunct to vitreoretinal surgery. There are also other sources of plasmin. Studies²⁵⁵ in Marburg, Germany, have shown that effective intravitreal levels of plasmin can be generated by injecting tissue plasminogen activator (tPA) and breaking down the blood–vitreous barrier with cryopexy or laser photocoagulation. Other studies²⁵⁶ have injected combinations of plasminogen and urokinase into the eye where they reacted with one another to generate plasmin. Recently, microplasmin, a human recombinant molecule with a molecular weight of 29,000 kd representing a portion of the plasmin molecule that lacks the five kringle domains but contains the protease domain of plasminogen, has been shown to separate the vitreous cortex from the retina in pigs²⁵⁷ and postmortem human eyes.²⁵⁸

Another relatively nonspecific protease currently under investigation is dispase. The first investigations²⁵⁹ to use dispase-induced PVD in enucleated porcine and human cadaver eyes, noting no untoward effects upon retinal histology and ultrastructure. Subsequent studies²⁶⁰ were successful in using this agent in vivo to remove cortical vitreous during vitreous surgery pigs. Because dispase has proteolytic activity against type IV collagen and fibronectin, there is some concern that the inner retina might be adversely affected by this agent. Yet, the histology in the aforementioned studies of porcine and human cadaver eyes found that only the lamina rara interna of the ILL was affected, with lesser effects upon the lamina densa and no effects upon neuroretinal ultrastructure. The in vivo animal studies also found that this agent did not alter the electroretinorgram. However, the most recent and extensive study²⁶¹ in rabbit and human eyes found that this agent causes retinal toxicity, perhaps due to the broad range of proteins subject to the effects of dispase.

REPLACING VITRECTOMY

In addition to facilitating vitreoretinal surgery as currently performed, pharmacologic vitreolysis could possibly replace vitrectomy, as was proposed with hyaluronidase to clear vitreous hemorrhage without vitrectomy. However, in the phase III Food and Drug Administration (FDA) trial undertaken in the United States, this drug was not found to be effective. This may be because the trial included both patients with type 1 and type 2 diabetes. In the former group, patients are younger and more likely to have an attached vitreous without a weakened vitreoretinal interface. Thus, although hyaluronidase may decrease the viscosity of gel vitreous and facilitate the outflow of red blood cells, it will not likely induce PVD. Thus, in the younger patients with type 1 diabetes the persistent attachment of the posterior vitreous cortex to the retina and to any neovascular complexes arising from the retina and optic disc would cause recurrent vitreous hemorrhage. The results of the phase III FDA trial would suggest that hyaluronidase is unable to induce PVD in diabetic retinopathy, consistent with the findings in animal studies.^262,263 On the other hand, combining hyaluronidase with SF₆ has purportedly induced PVD in the rabbit, similar to what has been shown with plasmin and ²⁶⁴ It is plausible that the expanding gas, and not the enzyme, is responsible for these effects, because many years ago Thresher, Ehrenberg and Machemer reported similar effects with expanding gas alone.²⁶⁵

PROPHYLACTIC POSTERIOR VITREOUS DETACHMENT

The true promise of pharmacologic vitreolysis lies in the potential to induce prophylactic PVD. To do so safely and effectively would require an agent that can liquefy the gel vitreous and also weaken vitreoretinal adhesion. To date, the leading candidate in the category of substrate-specific enzymes is chondroitinase. This agent lyses chondroitin sulfate, a molecule that appears to be important in both the maintenance of the gel state of vitreous (see above), as well as vitreoretinal adhesion. Experimental studies²⁶⁶ have shown that when used as an adjunct to vitreous surgery, chondroitinase facilitates the removal of premacular membranes. Several years ago, a phase I trial used this agent during vitreous surgery in patients with macular holes and proliferative diabetic retinopathy with no untoward effects. Phase II studies are being planned. However, the true value of this agent probably lies in its ability to induce both gel liquefaction and dehiscence at the vitreoretinal interface, making it an excellent candidate for prophylactic induction of PVD. In patients with advanced nonproliferative diabetic retinopathy who are at risk of progressing to proliferative retinopathy, liquefaction of the gel vitreous and detachment of the posterior vitreous cortex away from the retina prior to the onset of new vessel growth into the vitreoretinal interface will have a far better prognosis than if the vitreous were still attached to the retina. This may also mitigate against diabetic macular edema, a common cause of vision loss in diabetic patients. Fellow eyes of macular holes or retinal detachments, and indeed any manifestation of anomalous PVD (see above) would be suitable candidates for prophylactic pharmacologic vitreolysis.

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