Structural diversity in the gel/liquid proportions is exhibited in birds and fish, where the biophysical differences may relate to movements of the lens. In birds, where the changes in focal depth are manipulated by change in shape of the lens, the vitreous is more liquid. In contrast, where accommodation is accomplished by backward and forward movements, the material behind the lens is very viscous (Balazs 1960). A primary role of the vitreous humour is therefore a hydraulic damper, cushioning the lens during movements of the head and focusing. Other roles in helping to physically support the retina and having a nutritive role were not immediately apparent to early investigators. The connections into the anterior chamber via the porous hyaloid membrane and the ease of material exchange in both forward and backward directions relative to its position makes the vitreous humour an ideal reservoir for metabolic nutrients and a waste repository for the surrounding tissues. The transport processes within the vitreous cavity are closely regulated to maintain visual clarity, keeping the light path free from scattering, diffusing and absorbing components. It has also been described as a “sink” for some proteins and solutes, which are unable to cross over the blood–retinal barrier (Bito 1977).

6.2  Vitreous Anatomy

In the young, the vitreous humour is characterised as a flattened spherical body, indented by the lens. It is firmly attached to the retina in the anterior portion, in the region of the macular and optic nerve head. The volume is around 4 mL with variation in dynamic viscosity when sampled in different regions. Balaz has commented that the structure of the vitreous is so complicated, that no two sampled regions are the same. In cross-section, the points of attachment and gaps between vitreous and retina are clearly seen (Fig. 6.1). In the very young, the vitreous is adherent to the posterior surface of the lens, but after adolescence, a capillary channel appears allowing communication of solution between anterior and posterior regions of the anterior chamber (Kagemann et al. 2006). In modelling drug movement between vitreous and anterior chamber, the dimensions of the gap between the anterior boundary of the vitreous and the ciliary body, the retrozonulkar space of Petit, appears to be important in reconciling theoretical and actual data (Missel et al. 2010). Another important gap – that between vitreous and retina beyond the anterior points of attachment as illustrated in Fig. 6.1 – may be important in movement of molecules from the vitreous body.

The vitreous humour is composed of approximately 99% water but owes its viscoelastic properties to other components contained in the vitreous; these include collagen, hyaluronic acid and proteoglycans (Balazs and Denlinger 1984). Various types of collagen are present, with type II collagen being most predominant. The collagen fibres are arranged in a linear fashion with hyaluronic acid molecules dispersed in spaces between the fibres, trapping the water molecules (Sebag and Balazs 1989). The important role of the collagen is illustrated in genetic mutation. Where type II collagen is absent, as in Stickler Syndrome associated with a COL2A1 gene

Fig. 6.1  Main structures of the eye showing retina, vitreous humour and inner limiting membrane (adapted from N.E.I. source)

mutation, the eye exhibits high myopia; glaucoma may be evident and retinal detachment a significant risk (Richards et al. 2000).

The interactions between collagen and hyaluronan result in a lightly cross-linked polymeric meshwork. There is a higher abundance of collagen around the edge of the vitreous boundary, forming a more stretchable and rigid outer zone (Balazs 1960). Higher molecular weight hyaluronans can be found in greater concentration nearer to the lens, leading to higher viscosity at the anterior region and the lowest closer to the retina (Bettelheim and Samuel Zigler 2004). The structured network formed by collagen fibrils and hyaluronic acid results in a diffusion barrier to the entry of cells and macromolecules, whereas small molecules such as water and electrolytes can freely diffuse in all directions. The biochemistry and physicochemical properties of the medium has continued to interest, particularly with regard to vitreous gel replacement (Sebag 1998; Bishop 2000; Ciferri and Magnasco 2007).

Although the vitreous humour is avascular in nature, the circulation systems within its vicinity, including suprachoroidal and episcleral vascular currents, allow adequate drainage of materials injected or removal of metabolic wastes from the vitreous.

This group of external circulation systems may continuously clear drug substances introduced periocularly, resulting in poor penetration into the vitreous cavity thereby providing a considerable challenge in the use of topical, sub-tenons injection and transcleral modalities of drug delivery. A better understanding of the factors which influence drug distribution could potentially improve treatment options in degenerative diseases of the retina by enabling improved drug targeting to the desired site of action and allow prediction of toxicity. Currently, intravitreal drug administration appears to be the surest option in achieving therapeutic drug concentrations in the posterior eye, although issues of maintaining effective concentrations at the target remain.

6.2.1  The Inner Limiting Membrane

The inner limiting membrane of the retina is formed from components of the vitreous body and retina, and therefore forms a potential barrier for intra-ocularly injected drugs, except at the optic disc where it is absent. The ILM is between 1 and 3 mm thick and is composed of proteoglycans and type IV collagen.

The structure forms the basal lamellar of the Műller cells, which are glial cells funnelling the image projected onto the retina towards the photoreceptors; the Műller cell layer is therefore firmly anchored into the membrane. Halfter and colleagues have conducted studies on the embryonic development of the chick eye and speculate that the inner limiting membrane and vitreous body are needed during early maturation but can be dispensed with in later life (Halfter 1998). Early removal results in retinal dysfunction including massive loss of ganglion cells and retinal dysplasia, whereas later removal appears without effect and in some cases is useful. For example, on maturity, the remnants of epiretinal tissue (ERM) sitting on top of the ILM may lead to distortion of vision with a decrease in visual acuity. Vitreomacular traction has been described as a principle causative factor in the progress of diabetic macular oedema and staining with indocyanine green or infracyanine green to assist the peeling of the inner limiting membrane, is well established in macular hole surgery and has been investigated in DME (Kolancy et al. 2005) although the benefit of the procedure on quality of life may be modest in this disease compared to treatment of ERM (Okamoto et al. 2010).

Gauthier et al. have described adenovirus-mediated transfection (AAV) of Műller cells with brain-derived neurotrophic factor in Sprague–Dawley rats, dosing 5 mL into the vitreous chamber. The data obtained suggests that Műller cells are stimulated to produce factors which result in prolonged photoreceptor survival (Gauthier et al. 2005). Dalkara et al. have suggested that the inner limiting membrane is a barrier to some AAV serotypes and others, not expressing a suitable receptor, show no accumulation. Moreover, in those serotypes that show efficiency, the transduction is limited to the inner retina. The workers suggest that mild disruption with a protease might extend the progression of the transfection to deeper layers within the retina (Dalkara et al. 2009).

Fig. 6.2  Combined bright field and fluorescence micrographs after intravitreal injection of becavizumab, stained with Cy3-labelled donkey anti-human IgG. Part of an illustration from Heiduschka et al. (2007). Retinal pigment epithelium (RPE), photoreceptors of the outer layer (OLPR) and inner limiting membrane (ILM) identified. See text for further details (adapted from Heiduschka et al. 2007, with permission)

Heiduscka et al. conducted an examination of whether intravitreally injected Avastin (bevacizumab) would penetrate the retina of the cynomolgus monkey (Macaca fascicularis) following intravitreal injection. The animals were killed at 1, 4, 7 and 14 days post-injection and retinal slices prepared after fixing, embedding and staining. Figure 6.2 shows a selection from the images, which were stained with Cy3-labelled donkey anti-human IgG to detect the bevacizumab. The figures show a combined stain and phase contrast. On the first day, association with the inner limiting membrane is seen and residual staining of this layer at 7 and 14 days is evident. The material is transferred at an early stage to the choroid, and in the illustration, material in a choroidal vessel is identified. At 7 and 14 days, strong staining of the outer photoreceptor layer is noted, with the residual antibody remaining associated with the ILM.

Although the ILM appears to be a significant barrier as shown by the staining at later time points, material crosses the retina at an early stage post-injection, suggesting a shunt mechanism may operate. Wolter conducted examinations of eyes removed at surgery and noted the presence of pores in the internal limiting membrane of the normal human retina, located along the branches of retinal blood vessels (Wolter 1964). Microscopically, the breaks in the ILM are clearly seen (Fig. 6.3). It is suggested that these breaks allow for the migration of phagocytes and also microglia between retina and vitreous space. In the periphery of the normal retina of eyes of virtually all persons over 40 years of age, strands extend from the vitreous through the pores into the retina and surround blood vessels.