The Vitreous - Clinical Tree

Introduction

The vitreous body makes up approximately 80% of the volume of the eye and thus is the largest single structure of the eye ( Fig. 6.1 ). In the anterior segment of the eye, it is delineated by and adjoins the ciliary body, the zonules, and the lens. In the posterior segment of the eye, the vitreous body is delineated by and adjoins the retina.

Figure 6.1, Sketch of the primate eye showing the vitreous body and its relations. Wieger's ligament is the attachment of the vitreous to the lens. Berger's space and the Cloquet's canal are the former sites of the hyaloid artery.

The vitreous body has many normal physiological functions. This chapter focuses on the most important physiologic relationships, especially those that have a close clinical correlation. As background for the understanding of the physiology and the pathophysiology of the vitreous body, we focus on the main features of the anatomy, biochemistry, and biophysics.

The investigation of the vitreous body and its structure and function is hampered by two fundamental difficulties. Firstly any attempts to define vitreous morphology are in fact attempts to visualize a tissue, which by design is intended to be invisible. Secondly the various techniques that have previously been employed to define the structure of the vitreous body are combined with artifacts that make interpretations difficult in terms of the true in vivo physiological situation.

Anatomy

Embryology

Structural considerations of embryology

In the early stages the optic cup is mainly occupied by the lens vesicle. As the cup grows the space formed is filled by a system of fibrillar material, presumably secreted by the cells of the embryonic retina. Later, with the penetration of the hyaloid artery, more fibrillar material apparently originating from the cells of the wall of the artery and other vessels contribute to filling the space. The combined mass is known as primary vitreous .

The secondary vitreous develops later, appearing at the end of the sixth week, and is associated with the increasing size of the vitreous cavity and the regression of the hyaloid vascular system. The main hyaloid artery remains for some time, but it eventually disappears and leaves in its place a tube of primary vitreous surrounded by the secondary vitreous, running from the retrolental space to the optic nerve (area of Martegiani). The tube is called Cloquet's canal ( Fig. 6.1 ); this is not a liquid-filled canal, but simply a portion of differentiated gel devoid of collagen fibrils.

The term tertiary vitreous is related to the fibrillary material, which develops as the suspensor fibrils, the zonules, of the lens. During childhood the vitreous undergoes significant growth. The length of the vitreous body in the newborn eye is approximately 10.5 mm, and by the age of 13 years, the actual length of the vitreous increases to 16.1 mm in the male. In the absence of refractive changes, the mean adult vitreous is 16.5 mm.

Molecular and cellular considerations of embryology

The two main components of the vitreous, collagen and hyaluronic acid, are produced in the primary and secondary vitreous. In the primary vitreous, however, there is initial production of substances other than hyaluronic acid, such as galactosaminoglycans; later hyaluronic acid becomes the predominant constituent.

The primary vitreous contains cells which in the secondary vitreous differentiate as hyalocytes and fibroblasts. The hyalocytes are believed to be involved in the production of glycosaminoglycans, especially hyaluronic acid, a non-sulfated glycosaminoglycan.

Although the function of the fibroblasts is not known exactly, they are probably involved in the formation of collagen. The retina may also be a source of collagen synthesis. The hyalocytes are found in the vitreous cortex, approximately 30 µm from the internal limiting membrane (ILM), with the highest density near the vitreous base and the posterior pole.

Anatomy of the mature vitreous body

The mature vitreous body is a transparent gel which occupies the vitreous cavity. It has an almost spherical appearance, except for the anterior part, which is concave, corresponding to the presence of the crystallin lens. The vitreous body is a transparent gel; however, it is not completely homogeneous ( Fig. 6.2 ). The outermost part of the vitreous, called the cortex, is divided into an anterior cortex and a posterior cortex, the latter being approximately 100 µm thick ( Fig. 6.3 ). The cortex is also called the anterior and the posterior hyaloid . The cortex consists of densely packed collagen fibrils ( Fig. 6.4 ). The vitreous base (see Fig. 6.1 ) is a three-dimensional zone. It extends approximately from 2 mm anterior to the ora serrate to 3 mm posterior to the ora serrata, and it is several millimeters thick. The collagen fibrils are especially densely packed in this region.

Figure 6.2, Human vitreous dissection. ( A ) Vitreous of a 9-month-old child. The sclera, choroid and retina were dissected off the vitreous, which remains attached to the anterior segment. A band of gray tissue can be seen posterior to the ora serrata. This is peripheral retina that was firmly adherent to the posterior vitreous base and could not be dissected. The vitreous is solid and although situated on a surgical towel exposed to room air maintains its shape because at this age the vitreous is almost entirely gel. ( B ) Human vitreous dissected off the sclera, choroid and retina are still attached to the anterior segment. The specimen is mounted on a lucite frame using sutures through the limbus and is then immersed in a lucite chamber containing an isotonic, physiologic solution that maintains the turgescence of the vitreous and avoids collapse and artefactual distortion of vitreous structure.

Figure 6.3, Human vitreous structure during childhood. This view of the central vitreous from an 11-year-old child demonstrates a dense vitreous cortex with hyalocytes. The posterior aspect of the lens is seen below, though dimly illuminated. No fibers are present in the vitreous.

Figure 6.4, Ultrastructure of human vitreous cortex. Scanning electron microscopy demonstrates the dense packing of collagen fibrils in the vitreous cortex. To some extent this arrangement is exaggerated by the dehydration that occurs during specimen preparation for scanning electron microscopy (magnification = ×3750).

The vitreoretinal interface

The vitreoretinal interface can be defined from electron microscopy as the outer part of the vitreous cortex (posterior hyaloid), including anchoring fibrils of the vitreous body and the ILM of the retina ( Fig. 6.5 ). The ILM is a retinal structure between 1 and 3 µm thick, consisting mainly of type IV collagen and proteoglycans. It contains several layers and can be considered the basal lamina of the Müller cells, the foot processes of which are in close contact with the membrane.

The vitreous cortex is firmly attached to the ILM in the vitreous base region, around the optic disc (Weiss ring), at the vessels, and in the area surrounding the foveola at a diameter of 500 µm. Under normal conditions, the connection between the fibrils of vitreous cortex and the ILM is looser than in the rest of the vitreoretinal interface. The adhesion is strong in young individuals, and dissection of the retina from the vitreous often leaves ILM tissue adherent to the vitreous cortex. Under pathologic conditions, the tight connections between the vitreous cortex and the ILM play an important role, as is discussed later in this chapter.

Ultrastructural, biochemical, and biophysical aspects

Ultrastructural and biochemical aspects

The vitreous contains more than 99% water; the rest is composed of solids. The vitreous acts as a gel (i.e. an interconnected meshwork) that surrounds and stabilizes a large amount of water compared with the amount of solids. The gel structure of the vitreous results from the arrangement of long, thick, non-branching, collagen fibrils suspended in a network of hyaluronic acid, which stabilize the gel structure and the conformation of the collagen fibrils ( Figs 6.6 and 6.7 ).

Figure 6.6, Ultrastructure of hyaluronic acid/collagen interaction in the vitreous. Specimen was fixed in glutaraldehyde/paraformaldehyde and stained with ruthenium red. Collagen fibrils (C) are coated with amorphous material (A) believed to be hyaluronic acid. The amorphous material may connect to the collagen fibril via another glycosaminoglycan, possibly chondroitin sulfate (see inset). Interconnecting filaments (IF) appear to bridge between collagen fibrils, inserting or attaching at sites of hyaluronic acid adhesion to the collagen fibrils (bar = 0.1 µm).

Figure 6.7, Ultrastructure of human vitreous. ( A ) Specimens were centrifuged to concentrate structural elements, but contained no membranes or membranous structures. Only collagen fibrils were detected. There were also bundles of parallel collagen fibrils such as the one shown here in cross-section (arrow). ( B ) Schematic diagram of vitreous ultrastructure, depicting the dissociation of hyaluronic acid (HA) molecules and collagen fibrils. The fibrils aggregate into bundles of packed parallel units. The HA molecules fill the spaces between the packed collagen fibrils and form “channels” of liquid vitreous.

In the human eye the major part of the glycosaminoglycan is hyaluronic acid, with a molecular weight of 3–4.5 × 10 ⁶ . The volume of non-hydrated hyaluronic acid is 0.66 cm ³ /g, in contrast with the volume of the hydrated molecule, which is 2000–3000 cm ³ /g. The molecule forms into large, open coils, with the anionic sites spread apart. This arrangement of small-diameter fibers, separated by highly hydrated glycosaminoglycan chains, permits the transmission of light to the retina with minimal scattering. The collagen fibrils in the vitreous are thin, with diameters of approximately 10–20 nm. Collagen fibrils are mostly of collagen type II. They are composed of three identical α-chains, which form a triple helix. The helix is stabilized by hydrogen bonds between opposing residues in different chains. Collagen type IX is also present and may function as a bridge, linking type II collagen fibrils together. Collagen V/XI is integrated with collagen II in the collagen fibers. The collagen fibrils seem to interconnect with the hyaluronic acid, most likely via bridging glucoproteins. The viscoelastic properties of the vitreous gel are neither due to hyaluronic acid or collagen alone but to the combination of the two molecules.

Dissolved in the water of the vitreous gel are inorganic and organic substances as shown in Table 6.1 , where plasma values are given for comparison.

Table 6.1

Concentration of various substances in the vitreous (weighted averages in mmol/kg H ₂ O)

All other values are rabbit data from Reddy & Kinsey 1960; with modification from Kinsey 1967.

Inorganic substances
	Sodium	Potassium	Calcium	Magnesium	Chloride	Phosphate	pH
Vitreous	134	9.5	5.4 *	2.3 *	105	2	7.29 **
Plasma	143	5.6	9.9 *	2.2 *	97	0.4	7.41 **

Organic substances
	Ascorbate	Glucose	Lactate
Vitreous	0.46	3.0	12.0
Plasma	0.04	5.7	10.3

* Human data from McNeil et al 1999.

** Porcine data from Andersen 1991.

According to Table 6.1 , it appears that gradients exist in both directions between vitreous and plasma. These gradients are a result of several mechanisms: presence of the blood–ocular barriers (i.e. active and passive passage across the barriers), metabolism in retina and ciliary body, and diffusion processes in the vitreous body ( Box 6.1 ).

Box 6.1

Vitreous – aging and ocular pathology

The concentration of salts and organic substances of the vitreous differ substantially from plasma due to the blood–aqueous and blood–retinal barrier
Small molecules move through the vitreous gel by diffusion
Vitreous fluorometry is useful for evaluation of the vitreal morphology, the fluorescein profile is an indicator of physiologic aging such as vitreous liquefaction
The aging process leads to posterior vitreous detachment, easily visualized by optical coherence tomography (OCT)
Vitreoretinal traction may lead to formation of a macular hole and the traction can be conducted through the retinal layers
Vitreoretinal traction is also implicated in some cases of macular edema
In diabetes, the high glucose speeds up metabolism before visible retinopathy
Increased demand for oxygen and capillary closure leads to retinal ischemia and an increased production of VEGF
Increased leakage through the blood–retinal barrier leads to macular edema
VEGF inhibition and steroids decrease macular edema

The values in Table 6.1 represent mean values for the whole vitreous. The methods used to quantitate vitreous concentrations are difficult and may differ between studies in absolute numbers. However, regional differences within the vitreous have been measured for some substances. Figures 6.8 to 6.10 show the regional difference for glucose ( Fig. 6.8 ), lactate ( Fig. 6.9 ), and oxygen ( Fig. 6.10 ). The fall in vitreous oxygen tension towards the center, corresponding to the upper curve in Figure 6.10 , was also found by Sakaue and seems to result from an oxygen flux from the retina towards the vitreous corresponding to arterioles; the flux goes in the opposite direction corresponding to the venules (lower curve). Several studies have found an increase in preretinal oxygen after photocoagulation, indicating that the oxygen supply to the inner retina improves after destruction of the outer retina and a concomitant decrease in tissue metabolism and oxygen needs.

Figure 6.8, Glucose concentration in different parts of the vitreous body and in plasma. All values are in µmol/g tissue weight (mean ± standard deviation, n = 20).

Figure 6.9, Lactate concentration in different parts of the vitreous body and in plasma. All values are in µmol/g tissue weight (mean ± standard deviation, n = 20).

Figure 6.10, Heterogeneity of the P o 2 in the preretinal vitreous of a non-photocoagulated eye. Graphic representation of the P o 2 (± SEM) recorded when the O 2 -sensitive microelectrode was withdrawn from the vitreal surface of the retina (x = 0) towards the vitreous. Curve A: opposite an arteriole: curve I: opposite an intervascular zone: curve V: opposite a vein. These results are averages of measurements made on one or both eyes of 11 miniature pigs.

Biophysical aspects

The gel structure acts as a barrier against movement of solutes. Basically, substances may move by two different processes: diffusion or bulk flow. The diffusion process can be illustrated in humans by using fluorescein as a tracer substance for the biophysical behavior of the gel. The fluorescein concentration in the vitreous body can be estimated by vitreous fluorophotometry. After intravenous (IV) injection of fluorescein, a certain amount (in healthy humans only a very small amount) passes through the ocular barriers into the anterior chamber and into the vitreous body. The ILM, the vitreoretinal interface, and the vitreous cortex cannot be regarded as a diffusion restriction to smaller molecules ( Box 6.1 ). In the vitreous the distribution versus time occurs according to the diffusion properties of a particular molecule in the vitreous gel.

An analysis of the fluorescein concentration gradient in the posterior part of the vitreous can be made with the aid of a simplified mathematical model of the relationship between the vitreous body and the blood–retinal barrier, as shown in Figures 6.11–6.14 .

Figure 6.11, Simplified model of the eye used for the computerized calculation of a blood–retinal barrier permeability and vitreous body diffusion coefficient for the substance fluorescein. C o (t): concentration of free (not protein-bound) fluorescein in plasma at time (t): C (r,t): concentration of fluorescein in the vitreous body at time (t) and at the position (r) from the center of the eye. P: permeability of the blood–retinal barrier, symbolized by a single spherical shell: D: diffusion coefficient in the vitreous body.

Figure 6.12, Concentration of free (non-protein bound) fluorescein in ultrafiltrate of plasma versus time after IV injection of the dye. The data were obtained in a normal subject. Fluorescein was injected at time 0. The first blood sample was obtained at 5 min, then 15, 30, 60 and 120 min after the injection. The concentration during the first min of injection was not directly measured, but calculated (dotted line) – see method.

Figure 6.13, Vitreous fluorophotometry scan along the optical axis of the eye obtained 60 min after injection of fluorescein. The black arrow indicates the retina, the open arrow the fluorescein concentration in the anterior chamber. The autofluorescence signal from the lens has been removed. Note a small peak behind the lens (∼15 mm from the retina) due to fluorescein leaking from the anterior chamber into the vitreous body.

Figure 6.14, Diffusion coefficient for fluorescein in the vitreous body and fluorescein permeability of the blood–retinal barrier in diabetic patients with three different degrees of retinopathy.

In the model the vitreous body is considered as a globe with an outer delineation corresponding to the blood–retinal barrier ( Fig. 6.11 ). Fluorescein passes the barrier passively with permeability P. Diffusion in the vitreous gel takes place with a diffusion coefficient D. The time-dependent plasma fluorescein concentration is given by Co(t) and the concentration in the vitreous body dependent on time (t) and distance (r) from the center of the eye is given by C(r,t).

The basic equations and the mathematical formalisms are as follows:

$C (r, t) = \int_{0}^{t} C_{o} (t-s) * F (r, s; a, D, P) ds$

where

$F (r, s; a, D, P) = \frac{aP}{r \sqrt{D}} * [G (\frac{a - r}{2 \sqrt{D}}, s; k) - G (\frac{a + r}{2 \sqrt{D}}, s; k)]$

In equation (2) , G is given by

$G (x, s; k) = e^{- x2 / 4s} / \sqrt{π s} - {k * e}^{k (x + k * s)} erfc (k \sqrt{s} + x / 2 \sqrt{s})$

where
and erfc is the complementary error function. Radius [a] of the eye is determined experimentally. P and D are calculated from a set of experimental data by minimizing

$S = \sum_{i = 1}^{N} w_{i} {[C_{m} (r_{i}, t) - C (r_{i}, t)]}^{2}$

where C _m [r _i , t] is the measured value at r = r _i , and c is the corresponding value given by equation (1) .

Equation (4) indicates that each point of the vitreous concentration profile is weighed equally during the fitting procedure. In the paper by Larsen et al another weighing procedure was suggested. However, this procedure adds too much weight to the low values towards the center of the eye and, accordingly, the present equal weighting procedure is preferred.

Figure 6.12 shows the fluorescein concentration in the bloodstream versus time after IV injection. Figure 6.13 shows the fluorescein concentration in the vitreous body and the anterior chamber 60 minutes after the injection as determined by vitreous fluorophotometry. A combination of plasma and vitreous values by aid of the simplified mathematical model results by curve fitting in a diffusion coefficient of approximately 6 × 10 ⁻⁶ cm ² /sec. This is close to a diffusion coefficient that would be expected in an unstirred gel; experimentally, the diffusion coefficients for mannitol and inulin have been found to be 2.4 and 2.0 × 10 ⁻⁶ cm ² /sec respectively.

The diffusion coefficient for fluorescein in the vitreous in diabetic patients with different degrees of retinopathy is shown in Figure 6.14 . Although the permeability of the blood–retina barrier increases in relation to the degree of retinopathy, the diffusion coefficient is unchanged, indicating that the spread of fluorescein in the vitreous gel occurs with the same kinetics and rate during the earlier phases of diabetic retinopathy.

The permeability for the blood–retinal barrier relates to low-molecular-weight substances and ions; is low in the healthy eye, as shown here for fluorescein; and is close to the permeability of the blood–brain barrier. The blood–aqueous barrier (i.e. the barrier in the ciliary body and the iris) is looser, although it is still tighter than capillaries elsewhere, such as in the muscles.

The presence of the ocular barriers and the “slow” diffusion process have the consequence that transient changes in the bloodstream are reflected slowly in the total vitreous body ( Fig. 6.13 ). The slow change of the vitreous body concentration can be used in some aspects of legal medicine regarding postmortem diagnosis. The time constants for many substances ( Figs 6.15 and 6.16 ) are of the same magnitude as those describing glucose transport between blood and brain; that is, half of the maximum is achieved in approximately 10 minutes.

$Figure 6.15, Entry of 14 C-glucose from the blood into the aqueous (A.C.) and vitreous (V.C.) humors. Blood and aqueous humor samples were fractionated on Sephadex columns before counting to separate glucose from its metabolic products. Curves are hand-fitted to the data; 6 animals were used.$

Figure 6.16, Entry of H-lactate from the blood into the aqueous (A.C., anterior chamber; P.C., posterior chamber) and vitreous humors. The two lower curves in this figure are best fit lines obtained by analog computer analysis in accordance with the theory of aqueous humor dynamics derived by Kinsey & Reddy.

Bulk flow through the vitreous cavity as a result of a possible pressure gradient from the anterior part of the eye toward the posterior pole does not play any significant role in the distribution of low-molecular-weight substances in the intact vitreous; this aspect was not included in the mathematical model. However, high-molecular-weight substances or large particles are moving through the vitreous as a result of bulk flow (i.e. the flow of liquid that enters the vitreous body from the retrozonular space and leaves through the retina, as described by Fatt ). If such a compound is placed in the anterior part of the vitreous, it moves slowly toward the retina; the diffusion process is virtually zero for large molecules. In contrast, diffusion is more rapid for the movement of small molecules. Low-molecular-weight substances move faster, diffusing in all directions, and are virtually unaffected by bulk flow; if placed in the vitreous, a low-molecular-weight substance will also be found in the anterior chamber.

Aging of the vitreous

The vitreous body goes through considerable physiological changes during life; changes that have great significance for its function. There is a sliding transition between the physiological aging changes and actual degenerative changes (retinitis pigmentosa, Wagner's disease).

The normal postnatal vitreous body is a homogeneous gel developed and biochemically composed as described above. The fundamental aging change is a disintegration of the gel structure, the so-called liquefaction or synchysis, especially notable in the center of the vitreous where the collagen concentration is lowest. Liquefaction starts early in life, and a linear increase in the volume of vitreous liquid is found with age.

Molecular mechanisms in aging

The mechanisms behind the liquefaction are not known exactly but could be linked to conformational changes in the collagen. The apparent molecular weight of vitreous collagen increases with age because of the formation of new covalent cross links between the peptide chains equivalent to the aging process in collagen elsewhere in the body. Bundles of collagen fibrils become biomicroscopially visible as coarse fibrous opacities. The common aging processes, such as the cumulative effect of light exposure and non-enzymatic glycosylation, seem to be important. Both hyaluronic acid and collagen may be affected by free radicals in the presence of a photosensitizer such as riboflavin (which is present in the eye) after irradiation with white light. Enzymatic and non-enzymatic cross-linking have also been demonstrated. Non-enzymatic glycosylation is well known from other tissues with a slow turnover of proteins, such as the lens. Proteins are cross-linked due to the Maillard reaction with formation of a covalent binding between an amino group and glucose leading to insolubilized proteins (advanced glycation end-products – AGEs). The process is modulated by ultraviolet light and accelerated in persons with diabetes mellitus. The vitreous glucose concentration is doubled in persons with diabetes compared with that of healthy subjects. Sebag et al have found that collagens in the vitreous are cross-linked due to non-enzymatic glycolysation.

Other mechanisms are probably involved. The network density of collagen decreases in childhood due to the growth of the eye, which could destabilize the gel. On the other hand, the hyaluronic acid concentration is increased and leads to gel stabilization. The concentration of electrolytes, soluble protein and other substances such as metalloproteinase may change. The soluble protein concentration increases with age due to an increase in the leakage through the blood–retina barrier, which may play a role both in the normal aging process and in pathologic conditions such as diabetic retinopathy.

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