2.2.1 Outer Membranes; Conjunctiva, Cornea and Sclera

The internal components of the eye are held within tough membranes known as ‘tunica fibrosa oculi’; these tissues are subject to internal pressure of 13–19 mm Hg, necessary in maintaining the correct shape of the body. The outer membranes encase and protect its contents and consist of the transparent cornea (anterior portion, 17%), extending forwards relative to the main body of the organ, and the highly vasculated and fibrous opaque white sclera (posterior portion, 83%) [5]. A ring of tissue where the cornea and sclera meet is known as the limbus; here the cornea thickens before making the transition to the scleral membrane [10]. The limbus is populated with stem cells responsible for regeneration of the epithelium, necessary due to its fast cell turnover rate [1].

The cornea is a transparent avascular multilayered membrane (Figure 2.2) and historically five layers are described (epithelium, Bowman’s membrane, stroma, Descemet’s membrane and endothelium), each have specific properties. More recently, Dua et al. published a report of their discovery of an additional layer between the Descemet’s membrane and the stroma, named the ‘Dua’s layer’, which is a tough membrane ∼15 μm thick that is impervious to air [11].

The cornea is nourished by the aqueous humour from inside the eye and is cleansed, lubricated and oxygenated by mucus and the tear film at the outermost surface. Further metabolic support is provided from limbal capillary blood vessels, although these vessels do not normally spread into the cornea unless it becomes starved of oxygen for an extended length of time [1,5].

The epithelium is the external physical layer, a lipophilic tissue that offers around 90% impediment to hydrophilic drug penetration and 10% to hydrophobic drug formulations. The central region of the epithelium is ∼50 μm, thickening to ∼100 μm towards the limbus [12]. The epithelial membrane consists of three types of cells: basal, polygonal and squamous. New cells are generated from limbal stem cells at the basal layer, where they have column-like shape, as they develop; older cells are pushed forwards, changing shape in the process. When intact, the epithelium is highly impermeable to aqueous solutions due to its superficial layers having a closely packed order with gap and tight junctions that serve to prevent the ingress of foreign matter [13–15]. However, this barrier is compromised when the superficial cells that make up the epithelium are disrupted, although in a healthy eye an epithelial layer is quickly regenerated. Epithelial repair occurs initially by migration of cells from nearby regions, followed by a proliferative phase resulting in the eventual regeneration of normal epithelial structure [1]. The epithelial surface is further protected by a mucus layer anchored to microvilli attached to the superficial epithelial cells. This mucosal film affords an additional barrier function against foreign matter. The epithelial surface is protected, nourished, wetted and washed by a continuously replenished tear film, which also serves to smooth out any topographic irregularities, ensuring an optically clear window to transmit incident light towards the retina [5].

An important function of the epithelium that is often overlooked is the protection it offers against ultraviolet radiation. Epithelia have high concentrations of tryptophan residues and ascorbate, which are considered important in absorbing UVR [16]. Epithelial cells absorb UV to varying degrees depending on its intensity and wavelength. Cell apoptosis follows irradiation beyond the threshold limit and a maximum irradiance of 3.0 mW cm⁻², corresponding to a dose of 5.4 J cm⁻² at the corneal surface, is considered safe to prevent UVR penetration to the endothelium and beyond [17–19]. Endothelial cells are continuously regenerated, ensuring maintenance of this protective barrier. Deeper structures within the eye are safeguarded from UV damage due to its absorbance at the epithelium. Ultimately, cell death follows when exposure exceeds a threshold that can be tolerated. Rapid cell turnover ensures continuous replacement of older superficial epithelial cells. It is necessary to understand the importance of this function when faced with a treatment option that compromises this protective layer, for example, when considering photorefractive surgery (laser treatment) and corneal UV ‘A’ collagen cross-linking [20]. At the very least, measures must be taken to minimise exposure to strong light sources and daylight during the healing process; effective UV absorbing eyewear should be worn during this period [16–20].

The Bowman’s membrane forms a transitional layer towards the stroma. This structure is not considered to be a barrier to drug diffusion. It is a homogenous, acellular form of ∼8–14 μm thickness, this structure does not regenerate if it is damaged [8,10].

The stromal substantia propria makes up the main portion of the corneal thickness at around 90%; it consists of a hydrophilic gel made up of collagen fibrils, proteins and mucopolysaccharides, and contains between 75 and 80% water w/w. Consisting of lamellae packed loosely and parallel to the surface, interspersed with corneal corpuscles, a cell type that maintains this layer by generation of new collagen. The stroma is an aqueous environment through which hydrophilic compounds can readily diffuse. Due to its hydrophilic gel structure, the stroma is a resistant barrier to lipophilic compounds [5,8,10,21]. Further, it is an extremely sensitive tissue owing to a high density of nerves throughout [22]. In human corneas an extra membrane has recently been discovered by Dua et al., now known as the Dua’s layer [11].

Next there is another acellular layer, at around 6 μm thickness, the Descemet’s membrane is a tough homogenous and resilient membrane that supports the endothelium, which is also responsible for secreting this membrane. Normally the Descemet’s membrane is under tension due to pressure imposed by the aqueous humour; this force maintains the curvature of the cornea [5,8,10].

The innermost layer of the cornea is the endothelium, a single loose covering of flat epithelial like cells whose function is to allow the permeation of nutrients and to maintain the hydration of the stroma via a bicarbonate dependent Na⁺/K⁺ -ATPase pump. The correct level of hydration is important to maintain transparency of the cornea and to avoid oedema (swelling due to excess fluid) [8,10].

Maurice and Giardini measured the average thickness of human cornea and found it to be 0.5070 ± 0.0042 mm, determined from both eyes of 44 volunteers [23]. Whilst the mean value quoted by Bahr is given as 0.5650 ± 0.0042 mm, this study was based on a larger data set from 125 subjects [24].

Further protection is offered by a mucosal membrane, the conjunctiva. At the anterior surface of the eye is the bulbar. Here the membrane is transparent and avascular in the corneal region and loosely attached to the sclera beyond this. The vascularised palpebral conjunctiva lines the posterior surface of the eyelids. Between the bulbar and palpebral membrane there are loose bridges of tissue, known as the superior and inferior fornices, forming the conjunctival sac, which provides a convenient depository that can be exploited to act as a reservoir for instilled medication or the placement of drug loaded ocular insert [10,25]. Figure 2.3 shows conjunctival tissue of a human subject, clearly demonstrating the loose nature of tissue in this region, which allows the organ to move within the eye socket, and for the eyelids to close over the eye.

Around 60% of the eye’s refractive power is provided by the cornea; however, this refraction is fixed and the main focusing action of the eye is provided by the crystalline lens. The shape, hence focusing power of the lens, is controlled by the ciliary muscles. The lens hardens, becomes less elastic and takes on a yellow hue with advancing age; these effects result in a reduction of colour perception and ability of the lens to accommodate between near and far vision [5,26].

2.2.2 Aqueous Chamber, Lens and Vitreous Body

Immediately behind the cornea is found the anterior chamber; this structure houses the iris and aqueous humour, a clear aqueous fluid similar to blood plasma. With less protein, aqueous humour contains higher concentrations of ascorbate, pyruvate and lactate, and lower levels of glucose and urea than blood plasma. The aqueous humour maintains the pressure within the eye and is secreted into the posterior chamber by the ciliary body. Aqueous humour is continuously replenished, leaving the chamber via the trabecular meshwork and is drained through the canal of Schlemm [5,27]. Slight resistance offered by the drainage mechanisms ensures normal intraocular pressure is maintained at ∼16 mm Hg in a healthy subject; pressures greater than 21 mm Hg are associated with ocular hypertension. A hypertensive state can be painful and often leads to glaucoma, which can lead to permanent damage to the retina if left untreated [8]. Therapeutic treatments for ocular hypertension are mostly targeted at reducing production of aqueous humour, which subsequently leads to a reduction of pressure from within the eye [5].

Situated between the anterior chamber and the posterior chamber is the iris, which acts as a control to the intensity of light entering the eye; the iris also responds to our emotional state and vigilance. This component is part of the uveal tract, which also encompasses the choroid and ciliary body. These structures reside between the external ocular membranes and the retina in the posterior chamber, although it extends into the anterior chamber and can be seen as the coloured part of the eye [5]. The uveal tract is highly vasculated and is responsible for nourishment and metabolic processes. One of the more important functions of the uvea is to offer a ‘systemic to ocular barrier’ known as the blood–eye barrier (BEB), important in keeping the light sensitive pathways transparent [10].

The main body of the organ holds the vitreous humour occupying ∼80% of the internal volume of the eye at around 4 ml. An avascular and optically clear structure consisting of collagen, hyaluronic acid, polysaccharide and vitrosin, the largest component being water at ∼98%. Behaving similar to a gel-like body, molecular movement throughout this body is driven by diffusion rather than fluid movement. However, the vitreous humour becomes less viscous as a person ages and behaves more like a liquid than a gel at this stage. The vitreous humour keeps the retina in place against the choroid, a vascular membrane between the retina and the sclera [5,10,28].

Positioned between the aqueous chamber and vitreous body is the crystalline lens, which allows for accommodation between near and far vision. For distance vision the ciliary muscles relax and the lens becomes less convex. Conversely, for near vision the ciliary muscles ‘tense’ and the lens becomes more convex. The optical clarity of the lens is due to the precise alignment of a single cell type consisting of soluble protein fibres. It is contained within the capsular bag, which itself is attached to the ciliary body. Lens fibres are not regenerated if damaged and are, therefore, subject to age or disease-related degeneration; the structure becomes less elastic and takes on a yellow hue as a person becomes older, leading to less perception of colour and a gradual loss of the ability to accommodate between near and far vision. The most common cause of ‘loss of visual quality’ is disease causing the formation of cataracts [5,8,26,29].

2.2.3 Choroid and Retina

Known as the ‘tunica vasculosa oculi’, the choroid forms a vascular network around the back of the eye that includes the ‘iris’, ‘ciliary body’, covering the inside of the sclera between the sclera and retina. The main function is to provide nourishment to the retina, ciliary body and iris [27]. Membranes within the vascular network are special in that they offer a ‘blood–eye barrier’ to prevent ingress of substances that can compromise the clarity of the light transmitting pathways [10].

Adjacent to the choroid and kept in place by pressure from the vitreous body lies the ‘retina’, a membrane of highly specialised nerve tissue continuous to the optic nerve. It forms a link to the visual cortex, a specialised region of the brain dedicated to visual processing. The purpose of the retina is to convert light falling upon it into visual signals interpreted by the brain as images of the outside world [2]. The retina is a complex multilayer structure ∼0.5 mm thick lining the posterior inner surface of the eye, extending towards the ciliary body; the extent of coverage is shown in Figure 2.1. Specialised photoreceptor cells are embedded throughout the retina consisting of cones and rods. Cone cells are associated with colour perception, whilst rod cells detect light monochromatically and are sensitive even at low light levels; these are of particular importance in peripheral vision. There are three types of cone cell type acting as photoreceptive transducers employing the photosensitive molecule, rhodopsin, which respond to the primary colours, red, blue and green. Colour perception is mostly concentrated at or near the macula, whilst monochromatically sensitive rod cells are dispersed throughout the retina. Retinal nourishment and metabolic maintenance are regulated by the retinal pigmented epithelium (RPE), which itself is part of the blood–retina barrier (BRB); the RPE and BRB serve to regulate solute movement to the retina and into the body of the eye [5,28].