The cornea is a structure composed of several tissues, which responds to its particular location and functions. It is part of the wall of the eyeball and at the same time it is the first lens of the organ of vision. Therefore, it presents a series of characteristics such as its transparency, its mechanical resistance, its lack of vessels and its abundance of nerves, etc. Its external position makes it vulnerable to traumatisms and multiple types of insults, which, together with its peculiar physiology that limits its capacity of repair and its importance for vision, explain the need and importance of keratoplasties. On the other hand, that same composition by layers makes possible the lamellar approach for its replacement.

MACROSCOPIC ANATOMY

The cornea appears on external examination, as the first structure of the anterior segment (Figure 1). It has the shape of a spherical cap, with radii of anterior and posterior curvature of 7.8 and 6.5 mm on average, respectively. This makes it thinner in the center and causes some prominence with respect to the sclera, which defines a zone of inflection that, together with the loss of transparency, constitutes its limit or corneal limbus.

Its shape is elliptical with a horizontal axis about 1.1 mm greater than the vertical one – that is on average of 11.7 and 10.7 mm, respectively in men and women. This is because the opaque scleral tissue advances more over the limbus on the vertical axis; seen from the inside, the cornea is almost circular¹. From the refractive point of view, the cornea includes two approximately spherical dioptric surfaces, of convergent power of about 48.33 diopters (D) the anterior and divergent of about -6.15 D the posterior.

The cornea consists of five histological layers: the epithelium with its basement membrane, Bowman’s layer, the stroma, Descemet’s membrane and the endothelium (Figure 2). At present, the existence of a sixth one has been proposed: the predescemetic layer of Dua, which would be between the Descemet and the deep stroma. Although its practical value in surgery is undeniable, its existence as a distinct anatomical entity is controversial (see chapter 5.2).

THE TEAR FILM

Although the tear film is not normally counted among the histological layers of the cornea, its importance in corneal physiology makes it a crucial factor in the survival of keratoplasty. Its average thickness is usually estimated at around 7 mm², but it is variable and much higher values have been measured.

Its composition is complex: a superficial lipid layer, of mainly Meibomian origin and very thin (0.1 to 0.5 μm), limits evaporation, tear drop and external contamination. The main layer, aqueous or serous, secreted by the main and accessory lacrimal glands contains, in addition to water and electrolytes, numerous substances including some of defensive importance, such as lysozyme, lactoferrin and immunoglobulins. This layer is progressively mixed with a mucinous layer adhered to the epithelial surface and derived from the conjunctival goblet cells. The mucin allows the adhesion of the aqueous tear to the surface, otherwise hydrophobic, of the corneal epithelium (Figure 3).

THE CORNEAL EPITHELIUM

The corneal epithelium is a cellular tissue of high regenerative capacity and very impermeable to water-soluble substances, which makes it especially suitable for the defense of the cornea. Its thickness is about 50 μm at the center and consists of between 3 and 7 cellular layers, depending on the individuals and the area of the cornea (Figure 4). It is properly squamous in its superficial layers, while in its medium level the cells are polygonal and at their base they become cylindrical. The epithelium is constantly replaced (each cell in about 7 days)³ by desquamation of the superficial cells. Mitoses are produced from basal cells, which in turn are derived from limbal stem cells⁴.

The superficial or apical cells are flat and are characterized by the abundance of binding complexes. Its outer membrane is covered with microvilli as they mature, a roughness that helps to stabilize the tear film (Figure 5).

The polygonal or "winged" cells of the intermediate layers have large ovoid nuclei as well as abundant interdigitations and desmosomes. The basal cells are more elongated, and their content reveals their reproductive function rather than transport, in a relatively low aerobic environment: mitochondria abound less than in the endothelium and along with the prominent endoplasmic reticulum appear abundant granules of glycogen.

Although the epithelium plays a secondary role in the technical aspects of keratoplasty, it is essential for the survival of the grafts. The situations in which it is not possible to maintain a stable corneal epithelium, lead them to failure and are the subject of regenerative medicine techniques, or enter in the field of keratoprostheses.

SUBEPITHELIAL STRUCTURES

Between the epithelium and the stroma we find a series of specialized structures, which constitute the base on which the epithelium develops and probably contribute to the peculiar corneal biomechanics.

The epithelial basement membrane and anchoring structures

The corneal epithelium is maintained in its position thanks to its secreted basement membrane and anchoring structures (Figure 6). The basal cells are joined by hemidesmosomes to the basal membrane, which is composed of an anterior lucid lamina – about 23 nm thick – and a posterior dense lamina of about 48 nm⁵. Under this we find the reticular lamina – which belongs to the Bowman’s layer – formed by anchoring filaments of up to 2 μm in length and composed of collagen type VII, which end in deeper anchoring plates⁶.

The Bowman’s layer

It is an acellular sheet of 8 to 12 μm thick, possibly an anterior specialization of the corneal stroma. It appears almost exclusively in humans, primates and some birds, and is supposed to have a biomechanical reinforcement function. It consists of fluted fibers of collagen type I, with diameters of 20 to 25 nm and bands of 67 nm, randomly arranged in an amorphous matrix⁷. Its anterior limit is well defined under the basal epithelial membrane and is relatively smooth; not so much the posterior, where the stromal collagen fibrils can be contiguous or end up opening up in the amorphous substance. Even some fiber bundle of the middle stroma seems to be inserted obliquely in it, which explains the remarkable cohesion between both layers⁷.

THE CORNEAL STROMA

It is a dense connective tissue formed by collagen (especially type I), proteoglycans and relatively rare cells called keratocytes. It comprises the main part of the corneal thickness – about 0.45 mm in the center – and is arranged in several hundred lamellae, generally parallel to the corneal surface. The collagen fibrils appear to run without continuity until they become contiguous with the scleral fibrils in the limbus⁷.

The whole organization of the stroma seems highly regular, probably adapted for transparency. The type I collagen fibrils are striated as those of the Bowman layer (bands of 67 nm) but somewhat thicker (25-35 nm) and arranged neatly with interfibrillar spaces of 55-60 nm. This distance is much smaller than the critical dimension to create scattering of light (half the wavelength, that is 200-350 nm), and the difference in refractive indexes between the collagen and the fundamental substance would make the cornea opaque if it were not for a destructive interference effect that, thanks to the regularity of the fibrillar network, cancels out the scattered waves and allows transparency (Figure 7)⁸.

The organization of the stromal lamellae extends this regularity to a higher order, with the exceptions required by structural cohesion. The posterior lamellae are thicker and more organized than the anterior ones, and while the central ones tend to follow the vertical and horizontal axes⁹, the peripheral ones are more disorganized, and some are parallel to the limbus, although without forming a ring¹⁰. There are, however, bifurcations and some oblique beams that cross the lamellae and provide cohesive force¹¹.

The keratin-sulfate is the typical glycosaminoglycan (GAG) of the cornea. It predominates in the posterior and central regions, while the dermatan-sulfate does it in the anterior and in the periphery. Both bind to the collagen fibrils; the former absorbs and transfers water more easily than the latter, which has been related to the dynamics of corneal hydration as a function of the stromal depth¹². The different architecture of the lamellae and different composition of the posterior stroma with respect to the anterior one explains its different behavior, both in the case of lamellar surgical dissection and laser photodisruption.

Keratocytes are a special type of fibrocyte, with a stellate cell body (fusiform to the cut), large nucleus and relatively scarce cytoplasm with rough endoplasmic reticulum, free ribosomes and Golgi apparatus (Figure 7b). They are scattered among the stromal lamellae and fine processes that allow them to communicate. They are responsible for the maintenance of the stroma and, in response to the aggressions, take the form of fibroblasts, migrate to the margins of the wound and secrete collagen and glycoproteins, although without the original regular order.

DESCEMET’S MEMBRANE

It can be considered as the basal lamina of the endothelium – which produces it by successively adding layers throughout life –, which separates it from the stroma (Figure 8). Its thickness is about 3 μm at birth and 8-10 μm in the adult. Three layers can be distinguished: 1) a very thin or interfacial matrix (MIF) (0.3 μm), adjacent to the stromal interface, 2) the anterior with bands (2-4 μm) corresponding to the fetal portion, and 3) the posterior without bands (the rest), developed after birth (Figure 9). The main component is type IV collagen, although types VIII and III are also found, as well as V and VI in MIF¹³.

In front of Descemet's membrane would be the predescemetic layer of Dua, about 10 mm thick and acellular. This layer has been postulated to explain the behavior of the tissue during pneumodissection surgical maneuvers and, as mentioned, its histological nature is controversial (see chapter 5.2). The details of the ultrastructure of Descemet's membrane are also the subject of another chapter (see chapter 6.2).

The posterior layer without bands increases with age and becomes more than 2/3 of the total of Descemet’s: it constitutes a "historical record" of the physiological status of the endothelium. It grows more in women, becoming twice as much as in men around 70 years of age¹³. This can have consequences when visualizing it during the descemetorhexis or in the degree of winding of this sheet when it is isolated.

THE CORNEAL ENDOTHELIUM

It is a single layer of flat cells that form a typically hexagonal mosaic and covers the posterior aspect of the cornea (Figures 8A, 10). The endothelium regulates the hydration and nutrition of the cornea, allowing the nutrients to pass along with the water through relatively permeable intercellular spaces. The great hydrophilicity of the stromal GAGs explains the tendency to edema as soon as the endothelial ion pumping activity ceases, which returns electrolytes and water to the anterior chamber.

The morphology of the endothelium reflects these functions. The transport activity requires abundant ATP and therefore an aerobic metabolism. Therefore, mitochondria abound, along with the endoplasmic reticulum, smooth and rough, Golgi apparatus and a large central nucleus (Figure 9). When cut, the lateral cell walls are very tortuous, imbricating with the neighbors. Between the cells there are ion-permeable unions (gap junctions) and in the apical portion near the anterior chamber there are narrow junctions that do not completely seal this pathway but prevent the passage of macromolecules (> 80 kDa)¹⁴.

The endothelial population in humans apparently has no effective mitotic capacity and when cells are lost, the neighbors extend to cover the defect. Its density is maximum at birth, above 3,500 cells/mm²; it decreases first with corneal growth and then more slowly with age and external aggressions. Physiological cell loss has been estimated at 0.7% per year. The minimum to avoid edema would be between 400 and 700 cells/mm², although many corneal grafts remain transparent for many years with the endothelium in this range. A study in corneas of eye bank found in all ages (3 to 75 years) a clearly higher endothelial cell density in the periphery than in the center – an average of 3790 against 2463 cells/mm² –, where a reduction in the percentage of hexagons and an increase in the coefficient of variation of the cell area was observed. This suggests a possible peripheral area with regenerative capacity¹⁵.

CORNEAL INNERVATION

The cornea receives sensory innervation of the first branch (ophthalmic) of the trigeminal, especially through the long and short ciliary nerves. These run through the suprachoroid and form a perilimbic plexus, from which 60 to 70 radial branches split to penetrate into the middle corneal stroma. There are few branches towards the deep stroma and in humans nerve fibers have not been seen in the endothelium. On the contrary, abundant collaterals advance to form a plexus under Bowman's layer. After traversing it, they enter the epithelium forming complex free ends with up to several dozens of dubbed branches each one¹⁶ (Figure 11).

The corneal epithelium is one of the most densely innervated structures in the body, with up to 10,000 terminations per mm² and sensitivity several hundred times greater than that of the skin. This seems to be due to the need to maintain the tear film by detecting the break-up points. On the other hand, the innervation exerts on the epithelium neurotrophic regulation mechanisms, in which the sympathetic innervation, coming from superior cervical ganglion neurons, also influences¹⁷.