Cornea and Sclera - Clinical Tree

Introduction

The outermost, fibrous tunic of the human eye is the cornea and the sclera ( Fig. 4.1A,B ). Both are soft connective tissues designed to provide structural integrity of the globe and to protect the inner components of the eye from physical injury. The clear, transparent cornea ( Fig. 4.1A,C ) covers the anterior 1/6th of the total surface area of the globe, while the white, opaque sclera ( Fig. 4.1A ) covers the remaining 5/6ths. The cornea and the lens are the eye's primary refractive structures and both have two key optical properties to this end – refractive power (light refraction) and transparency (light transmission). The presence of a healthy cornea is essential for good vision as it is basically the window of the eye. The cornea is most analogous to the external lens of a compound lens camera. By comparison, the sclera predominantly serves more of a biomechanical function and is analogous to the housing of the camera and lens.

Figure 4.1, ( A ) Diffuse illumination slit-lamp image of the human cornea and sclera. ( B ) Main anatomical components of the globe with detailed emphasis on the corneal and scleral components. ( C ) Slit-beam illumination slit-lamp image of the human cornea shows an optical section of the tissue. Notice the slight light scattering that occurs in the tissue, mainly from cellular components in cornea. ( D ) Histologic section of the human cornea labeling the five main cellular and extracellular matrix layers (toluidine blue ×25).

The cornea is 540 to 700 µm thick and is arranged in five basic layers – epithelium, Bowman's layer, stroma proper, Descemet's membrane, and endothelium ( Fig. 4.1D ); each having distinctly different structural and functional charactistics. It also is composed of three major cell types – epithelial, stromal keratocyte, and endothelial cells. Two of these, epithelium and endothelium, form cellular barrier layers to the stroma. Thus, their resistance to diffusion of solutes and bulk fluid flow are of considerable importance to maintaining normal corneal function (resistance to diffusion of solutes and fluid flow: epithelium [2000] ≫ endothelium [10] > stroma [1]). All 3 can replicate through mitosis, but vary considerably in their in vivo proliferative capacity with epithelial cells having the highest rates of cell division and endothelial cells being the least renewable. This fact is seen clinically since epithelial cells can completely regenerate after injury (e.g. corneal abrasions), while endothelial cells, as a result of limited in vivo proliferation, are most commonly involved in age-related (e.g. Fuchs' dystrophy) or injury-related disease (e.g. pseudophakic bullous keratopathy – PBK) ultimately resulting in corneal edema and bullous keratopathy. Corneal stromal keratocytes are a middle ground compromise between these two extremes. One major disadvantage of the epithelium's high proliferative potential is that it can occasionally go unchecked, resulting in cancer (e.g. squamous cell cancers of the cornea), whereas keratocytes and endothelial cells do not have this risk.

The sclera is 0.3 to 1.35 mm thick and is arranged into three layers – episclera, scleral stroma proper, and lamina fusca; each having distinctly different structural and functional characteristics. The sclera is composed of only one major cell type, the sclera fibrocyte, which has moderate proliferative potential, like that of the corneal stromal keratoctye. As the sclera has no cellular barrier layers, its permeability properties are quite similar to that of the corneal stroma. The sclera is an excellent example of a tissue made for biomechanical stability as it is stiff, strong, and tough. As such, disease of the sclera commonly results in loss of tectonic support, abnormal size due to dysregulated growth as well as inflammatory conditions that commonly also affect the joints in the body. Many animals have a very rigid sclera that is often supported by bone or cartilage. Humans deviate from this extreme in that their sclera is a less rigid fibrous connective tissue, perhaps reflecting its need to maintain an even blood flow to the choroid and retina during large excursions in eye motility.

This chapter reviews and explains the structure and function of the cornea and sclera, providing a framework for understanding normal health and disease of each tissue with an emphasis on function.

Cornea

Embryology, growth, development, and aging

The foundations of contemporary corneal embryology and development stem from pioneering embryonic chick studies. Since few such detailed studies have been carried out in primates or other mammals, the information that follows has potential gaps in knowledge since species-related differences may exist. Following lens vesicle formation between 4 to 5 weeks' gestation (27–36 days), surface ectodermal cells cover the defect left by lens vesicle invagination and become the primitive, undifferentiated corneal epithelium, composed initially of two cell layers. Similar to the avian cornea, the primitive corneal epithelium of primates and higher mammals immediately produces a primary acellular corneal stroma, or post-epithelial layer. In primates, this is seen as the gradual subepithelial addition of diagonal and then randomly oriented fibrillar elements, which later thicken into collagen fibrils that are slightly smaller in diameter than stromal collagen fibrils. The Bowman's layer is thought to be a distinct, dense anterior-most remnant of this embryologic layer, which is first detectable by light microscopy around 20 weeks' gestation. In contrast to humans, lower mammals, such as rabbits and rodents, have an underdeveloped primary acellular corneal stroma and its residual remnant, the Bowman's layer, is indistinct and rather thin. Around 12 weeks' gestation, a time period between eyelid fusion at 8 weeks' gestation and eyelid opening at 26 weeks' gestation, the epithelium differentiates to become a stratified, squamous epithelium 4 cell layers thick, which then produces an epithelial basement membrane. It remains 4 cell layers thick until approximately 6 months after birth when it reaches adult levels of 4–6 cell layers thick. Early in gestation, the epithelial basement membrane and anchoring complexes on the basal surface of the epithelium are absent. Rudimentary epithelial basement membrane and anchoring complexes only become detectable by 17 weeks of gestation. With further development in utero, the thickness and number of these structures gradually increases.

A first wave of neural crest-derived mesenchymal cells begins to extend beneath the corneal epithelial cells from the limbus around 5 weeks' gestation (33 days); these cells form the primitive endothelium. The primitive endothelium is initially composed of two-cell layers. By 8 weeks' gestation, it becomes a monolayer that starts to produce Descemet's membrane, which becomes recognizable on light microscopy at 3–4 months' gestation. The epithelium and endothelium remain closely opposed until 7 weeks' gestation (49 days), when a second wave of mesenchymal cells begins to migrate centrally from the limbus between the epithelium and endothelium invading below and into most of the primary acellular stroma. The cells do not enter the anterior-most 10 µm of stroma, which lacks keratocan, a proteoglycan core protein signal thought to be required for cellular invasion. This second wave of cells forms the stroma proper, or secondary cellular corneal stroma, as production of lamellar collagen begins within a few days in a posterior-to-anterior fashion. It is believed that the invading mesenchymal cells, destined to become keratocytes, use the primary acellular stroma as a scaffold, primarily in the anterior third of stroma proper. This concurs with the significant lamellar interweaving and oblique lamellar orientation in the anterior third of post-natal human corneal stroma as well as the fact that each successive lamellar layer is rotated 1–2 degrees clockwise. This directional rotation is the same in both right and left eyes. In the posterior two-thirds of the stroma, the corneal stroma is composed of essentially orthogonal lamellae. By 3 months' gestation, corneal nerves invade the stroma and eventually penetrate through the Bowman's layer so that nerve endings develop in the epithelium. Studies also suggest that by 5 months' gestation, tight junctions form around all the corneal endothelial cells and, by 5 to 7 months' gestation, the in utero cornea becomes transparent as the density of functioning endothelial Na ⁺ /K ⁺ -ATPase metabolic pump sites increase to adult levels. By 7 months' gestation, the cornea resembles that of the adult in most structural characteristics other than size. At birth in the full-term infant, the horizontal corneal diameter is about 9.8 mm and the corneal surface area is around 102 mm ² . The cornea of the newborn infant is approximately 75–80 percent of the size of the adult human cornea ( Fig. 4.2A,B,C ), while the posterior segment is < 50 percent of adult size ( Fig. 4.2D ). At birth, the cross-sectional thickness of the 4-cell layer epithelium averages 50 µm, the Bowman's layer averages 10 µm, the central stroma proper averages 500 µm, Descemet's membrane averages 4 µm, and the endothelium averages 6 µm thick (total mean central corneal thickness ∼ 570 µm).

Figure 4.2, ( A ) Keratometry values plotted with respect to age on a logarithmic scale. Negative numbers represent months of prematurity; dot = mean value for each age group and bar = standard deviation.

During infancy, the cornea continues to grow, reaching adult size around 2 years of age with a horizontal diameter of 11.7 mm, surface area of 138 mm ² , anterior surface curvature of 44.1 D ( Fig. 4.2A ), and mean central corneal thickness of 544 µm ( Fig. 4.2C ). Thereafter, it changes very little in size, shape, transparency, or curvature, although a shift from with-the-rule to against-the-rule astigmatism has been associated with aging ( Fig. 4.2B ). Post-natal aging is associated with several structural changes to the corneal tissue including: (1) epithelial basement membrane growth or thickening of an additional 100 to 300 nm or a rate of approximately 30 nm per decade of life; (2) decreased keratocyte, sub-basal nerve fiber, and endothelial cell density, presumably from stress-induced premature senescence; (3) increased stiffness, strength, and toughness of the stroma, from enzymatic maturation- and non-enzymatic age-related glycation-induced cross-linking of collagen fibrils; (4) Descemet's membrane thickening of an additional 6–11 µm or a rate of approximately 1 µm per decade of life; and (5) possible degeneration of extracellular matrix structures. These structural and cellular changes, however, minimally affect the optical and barrier functions of the cornea and perhaps improve the mechanical function. For example, corneal ectasia from natural causes, like keratoconus, is rarely seen after 40 years of age. Only three documented negative functional consequences are associated with aging – impaired corneal wound healing, decreased corneal sensation, and decreased extensibility of its tissue. In elderly individuals or younger individuals with lipid abnormalities, the cornea often becomes yellowish in the periphery of the cornea due to a fine deposition of lipid. This condition is called arcus senilis.

Major corneal reference points and measurements

When viewed anteriorly in the living eye, the adult human cornea appears elliptical ( Fig. 4.3A ) as the largest diameter is typically in the horizontal meridian (mean 11.7 mm) and the smallest is in the vertical meridian (mean 10.6 mm). This elliptical configuration is brought about by anterior extension of the opaque sclera superiorly and inferiorly. When viewed from the posterior surface, the cornea is actually circular ( Fig. 4.3A ), with an average horizontal and vertical diameter of 11.7 mm. The average radius of curvature of the anterior and posterior corneal surface is 7.8 mm and 6.5 mm, respectively, which is significantly less than the 11.5 mm average radius of curvature of the sclera. This results in a small 1.5–2 mm transition zone that forms an external and internal surface groove, or scleral sulcus, where the steeper cornea meets the flatter sclera ( Fig. 4.3A ). This sulcus typically is not obvious clinically because it is filled in by overlying episclera and conjunctiva externally. The tissue in this transition zone is known as the limbus ( Fig. 4.3B ), which averages 1.5 mm wide in the horizontal meridian and 2 mm wide in the vertical meridian. It is important because it contains adult corneal stem cell populations, contains the trabecular meshwork, which is the conventional outflow pathway for the aqueous humor, and is the inciting site of pathology in a few immunologic diseases. The limbus is also a major anatomic reference point for planning surgical entry into the anterior segment because it appears clinically as a blue transition zone. Therefore, an incision placed anterior to the blue zone is anatomically in the peripheral cornea, safely inside the trabecular meshwork and stem cells. The cornea is thinner in the center, measuring on average 544 ± 34 µm (range: 440–650 µm) with ultrasound pachymetry, and increases in thickness in the periphery to approximately 700 µm as it reaches the limbus. A meta-analysis of all cross-sectional and longitudinal corneal thickness studies over a 30-year time period showed that no significant age-related change in central corneal thickness occurred beyond the infant years.

Figure 4.3, ( A ) Coronal views show the elliptical shape of the right cornea when viewed anteriorly (upper left) and the circular shape when viewed posteriorly (lower left). Superior axial view (right) illustrates how the right globe deviates from a perfect sphere. Dashed lines = theoretical spherical globe; solid lines = actual contour of the globe. ES = external sulcus; TB = temporal bulge.

The central cornea overlying the entrance pupil, which is a virtual image of the real pupil and is typically located 0.5 mm anterior to and 14 percent larger than the real pupil, contains the central or effective optical zone of the cornea ( Fig. 4.3C ). The central optical zone is the portion of the cornea that can successfully refract a cone-like bundle of light from a distant or near fixation target through the pupil of the eye and then directly refracts it onto the fovea. The central location and size of this central optical zone dynamically changes according to the location of the fixation target in relation to the cornea (e.g. more distant fixation target = larger-diameter central optical zone, off-center fixation target = off-center central optical zone) and the aperture of the real pupil in various lighting conditions since the bundle of light has the same cross-sectional shape (e.g. an oval pupil results in an oval central optical zone) as the pupil and its diameter are defined by the pupil's diameter as well as the location of fixation target. The central optical zone's diameter typically averages 3.6 ± 0.8 mm in photopic lighting and 5.8 ± 0.9 mm in scotopic light with a range between 1.5 to 9.0 mm depending on the lighting condition. The remaining cornea peripheral to this central optical zone is the peripheral optical zone, which can refract light through the entrance pupil, but it does so at such an acute angle that it only affects the more peripheral aspects of the retina including the macula ( Fig. 4.3C ); it rarely directly impacts foveal vision.

Within the central optical zone are three major corneal reference points ( Fig. 4.3D ) that are extremely useful in determining the shape, refractive power, and biomechanical properties of the cornea, since they are statically fixed reference points. The first is called the corneal apex; it is defined as the steepest point or area of the cornea. It typically is measured using a corneal keratographer, topographer, or tomographer and thus its exact location on the cornea is referenced in relation to the corneal intercept of the imaging device's optical axis ( Fig. 4.3E ). The corneal intercept of the imaging device's optical axis is termed the device's axis point (DAP). On average, the corneal apex usually is located 0.8 mm temporal and 0.2 mm superior to the DAP or 0.5 mm temporal and 0.5 mm superior to the corneal intercept of the line of sight of the eye ( Fig. 4.3D ), but considerable inter-individual variability is found in the location of the corneal apex in comparison to the average. The clinical utility of the corneal apex is that it is of paramount importance in the selection and fitting of contact lenses and in determining the geometric aspheric shape of the cornea. Significant decentration of the corneal apex from the DAP may give a false interpretation of corneal shape (e.g. asymmetric shape). The easiest way to clarify whether this is real or due to artifact is to aim the imaging device's optical axis directly at the corneal apex, which is more cumbersome and difficult to do than standard alignment position.

The second major corneal reference point is the corneal intercept of the line of sight, also known as the corneal sighting center (CSC). The line of sight is an actual principal axis of the real eye as opposed to a theoretical construct of a schematic eye (e.g. visual axis) and it is defined as the principal axis joining a distant fixation point to the fovea. It theoretically is always thought to cross the center of the entrance pupil, which may not be true in the real eye since it is known that the center of the entrance pupil dynamically and unpredictably shifts up to 0.7 mm in direction with changes in pupil diameter, whereas the line of sight is a statically fixed axis line. Thus, it perhaps is best thought of as a line connecting the fixation target to the CSC on the anterior surface of the cornea and then via an unknown non-linear pathway is refracted in the cornea and by the lens to focus on the fovea. The location of the line of sight and CSC are of utmost importance to know in certain clinical situations for getting the best visual results with keratorefractive surgery, particularly with retreatments and customized ablations, and for calculating the proper posterior chamber intraocular lens (PCIOL) power to put in during cataract extraction (CE), especially after previous refractive surgery or with anterior surface irregularities, since subclinical decentrations ≥ 0.5 mm or torsional misalignments ≥ 15° can yield unwanted visual symptoms (e.g. coma or other higher-order aberrations [HOAs]). On average, the CSC is 0.4 mm nasal and 0.3 mm superior to the dynamically, unfixed pupillary axis or 0.5 mm inferior and 0.5 mm nasal to the static, fixed corneal apex reference point ( Fig. 4.3D ), but its location overall is highly variable between different individuals. Using a keratographer, topographer, or tomographer in non-standard alignment position, the CSC can be directly determined by having the patient directly look at a luminous fixation point centered in the circular rings of the imaging device and then it is marked in reference to the center of the operator's screen. Because of practical difficulties in non-standard alignment, some clinicians approximate the CSC's position using the coaxially sighted corneal reflex, where the patient looks directly at a luminous fixation point centered in the circular rings of the imaging device and the anterior corneal surface's first Purkinje image is used to approximate the location of the CSC. This approximation method reportedly locates a point on average 0.02 ± 0.17 mm (range: −0.43 mm to +0.68 mm) from the actual CSC in normal eyes; this, however, may not be the case in diseased or surgically altered corneas.

The third major and newest corneal reference point is called the thinnest corneal point (TCP), defined as the thinnest point or zone of the entire cornea. It is measured using various tomography instruments, which enable a mathematical 3D reconstruction of the in vivo pachymetric distribution map, allowing one to evaluate the spatial variation of the thickness profile over the entire cornea. In the normal cornea, the average location and value of the TCP is 0.4 mm inferior and 0.4 mm temporal to CSC and 5 µm less thicker than the central corneal thickness at the CSC. The location and value of TCP is important because it allows clinical differentiation of corneas with normal biomechanical properties from those with current or previous keratectasia since no normal cornea was found in a group with a TCP more than 1 mm distance from the CSC and less than 500 µm of thickness.

If the central optical zone of the anterior corneal surface is regular, yet not uniformly spherical in each meridian, the condition of astigmatism usually results. With astigmatism, a distant fixation point is refracted by the cornea and lens to become two focal lines rather than a sharp image point. On the other hand, if the central optical zone is irregular, then irregular astigmatism usually results. In adult humans, the conjunctival surface area has been measured at approximately 17.65 cm ² and the corneal surface area measures 1.38 cm ² , giving a conjunctival-to-corneal surface area ratio of 12.8, which is important for drug delivery calculations.

Optical properties

Light refraction

The main optical measurements that determine the total refractive power of the eye are the anterior and posterior curvatures of both the central cornea and lens, the depth of the anterior chamber, and the axial length of the eye ( Fig. 4.4A ). As this chapter is strictly about the cornea and sclera, we will focus only on the optical properties of the cornea. Refractive power and aberrations induced by the optics of the cornea are primarily due to corneal curvature and contour, respectively. Both are descriptors of corneal shape. The contour of the anterior corneal surface is basically of an aspheric geometry with the corneal apex defining the point of greatest refractive power or steepest curvature and then it gradually and variably flattens from the apex to the periphery. Corneal asphericity has been known for over 100 years and has been modeled by various mathematical formulas in order to derive a quantitative approximation of contour. The central optical zone of the anterior corneal surface best corresponds to that of a conic section using the following formula, which requires only knowing two conic fit parameters – Q and R :

Q=p−1=(ba)2−1=Ra−1=(1−e2)−1 $Q = p - 1 = {(\frac{b}{a})}^{2} - 1 = \frac{R}{a} - 1 = (1 - e^{2}) - 1$

$Figure 4.4, ( A ) Diagram of the schematic eye with representative average dimensions in millimeters and refractive indices of the relaxed, non-accommodating eye. The starred values change with accommodation. These dimensions are average values used to construct a representative or schematic eye – in real life, all dimensions of the eye can vary greatly between individuals and from these average values. Upper-right diagram shows that the principle of corneal contact lens wear is to make the anterior corneal surface ineffective as it is now bathed with aqueous tears and the new anterior refractive surface is the air–anterior contact lens interface. Middle-right diagram shows that the principle of myopic correction using the excimer laser is based on a graded removal of central anterior corneal tissue to decrease the central anterior corneal curvature, analogous to the removal of a biologic contact lens that is thicker in the center. Lower-right diagram shows that the principle of hyperopic correction using the excimer laser is based on a graded removal of a peripheral and paracentral concave lenticule of tissue to increase the central anterior corneal curvature (i.e. a donut-shaped trough) analogous to the removal of a biologic contact that is thicker in the periphery and of no or minimal thickness in the center. ( B ) Variations in conicoid shape for different asphericities, or Q-factors, with the same radius of curvature. ( C ) Schematic diagrams showing the three possible anterior corneal surface contours with ray tracings from a distant fixation point being refracted onto the retina. Upper left diagram is of a prolate cornea (Q factor < 0), which has a larger peripheral radius of curvature than at the apex (R p > R a ). Lower middle diagram is of a spherical cornea (Q factor = 0), which has equal peripheral and apical radii of curvature (R a = R p ). Upper right diagram is of an oblate cornea (Q factor > 0), which has a smaller peripheral radius of curvature than at the apex (R a > R p ). As a prolate cornea reduces spherical aberrations, its image is more tightly or precisely focused than spherical or lastly oblate corneas. The mean value of the Q factor for normal healthy adult corneas is −0.2, which reduces natural spherical aberration by about half; a Q factor of −0.50 eliminates all spherical aberration. Thus, the human cornea typically is not designed to induce zero spherical aberrations. In fact, the human lens is optically coupled to cornea in such a way that spherical aberrations are reduced close to zero in youth since during childhood and young adulthood the lens has negative asphericity. Lens asphericity dynamically changes with aging, usually resulting in a Q of zero around age 40 and then has positive asphericity after age 40. The overall effect on the optical properties of the human eye is that it gains progressively more spherical and other optical aberrations with increasing age, which perhaps best explains the direct association of deterioration of visual performance and quality with aging. Most degradation of image quality is due to age-related changes in the lens.$

Q is a unitless asphericity factor or expression of the rate of curvature change from the apex of the cornea to the periphery ( Fig. 4.4B ); p is a geometric form factor; a and b are horizontal and vertical semi-meridian hemi-axes; R is the apical radius of curvature; and e is the eccentricity. Q averages −0.4 in early childhood, but then gets gradually slightly less negative with age such that the central optical zone has a mean Q of −0.2 in adulthood (range: −0.81 to +0.47). Q < 0 describes a prolate contour where the rate of curvature change from the apex is less than that of a sphere ( Fig. 4.4C ); most normal corneas are prolate as it is advantageous in that it compensates for spherical aberrations induced by larger pupil sizes, which project misaligned peripheral rays of light on the fovea. Q = 0 describes a spherical contour where the rate of curvature change from the apex to the periphery is zero, while Q > 0 describes an oblate contour where the rate of curvature change from the apex is more than that of a sphere ( Fig. 4.4C ). Oblate contours are typically present in ≤ 20 percent of the normal population. Interestingly, asphericity can significantly change after surgery, especially excimer laser keratorefractive surgery, usually resulting in various oblate contours. Although the contour of the central optical zone of the anterior corneal surface is the most important for directly impacting foveal vision and on-axis aberrations (spherical aberrations, coma, and other HOAs), recent study has also shown that the peripheral optical zone is important for off-axis aberrations (e.g. glare, halos, starbursts). This peripheral optical zone does not fit a conic section well, but rather fits a ninth-order polynomial formula best and has a measured Q of −0.4 in adulthood when the central 10 mm of cornea is best fit to this formula. A few reports on the posterior corneal surface suggest it also has a prolate contour too with a Q of −0.4, but its contribution to the total optical aberrations of the eye are less well known.

The actual total corneal dioptric power of the central 4.0 mm of cornea reportedly averages 42.4 ± 1.5 D (range: 38.4–46.3) of the eye's total dioptric power of 60 D. The location of the corneal apex compared to the CSC (generally less than 1 mm from the CSC and on average 0.7 D steeper than that at the CSC), the degree of asphericity of the anterior corneal surface, and the degree of anterior corneal surface-to-posterior corneal surface ratios can vary widely from one individual to another or even change with age in an individual. For these reasons, it is difficult to take these general population-averaged results as an empirically useful value. An individual's total corneal power along the line of sight of the eye should probably best be measured using the Gaussian optics formula:

$P_{t o t a l c o r n e a} = \frac{n_{c} - n_{a i r}}{r_{a n t}} + \frac{n_{a} - n_{c}}{r_{p o s t}} - (\frac{d}{n_{c}} \times (\frac{n_{c} - n_{a i r}}{r_{a n t}}) \times (\frac{n_{a} - n_{c}}{r_{p o s t}}))$

Where P _totalcornea equals diopters of optical power; n _air , n _c , and n _a are the indices of refraction in air (1.000), cornea (1.376), and aqueous humor (1.336), respectively; r _ant and r _post are the radii of curvature of the anterior (0.0078) and posterior (0.0065) corneal surface in meters, respectively; and d is the central corneal thickness (0.00054) in meters.

$\begin{matrix} 42.18 D = & \frac{1.376 - 1}{0.0078} + \frac{1.336 - 1.376}{0.0065} - \\ (\frac{0.00054}{1.376} \times (\frac{1.376 - 1}{0.0078}) \times (\frac{1.336 - 1.376}{0.0065})) \end{matrix}$

Therefore, the calculated total optical power of the cornea using known average major reference values is 48.21 − 6.15 + 0.12 = 42.18 D, which agrees closely with the actual mean of 42.4 D found in the study above.

Because the cornea is thinner in the center than in the periphery, it should act as a minus lens, but functions as a plus lens because the aqueous humor neutralizes most of the minus optical power on the posterior corneal surface. If we compute the power of the posterior corneal surface in air, we find the following:

$P_{p o s t c o r n e a} = \frac{n_{a} - n_{c}}{r_{p o s t}} = \frac{1 - 1.376}{0.0065} = - 57.85$

The resulting calculated total optical power of the cornea would then be 48.21 − 57.85 + 1.12 = −8.52 D, which is a minus lens.

From the foregoing calculations, it is obvious that the most important refractive surface for humans is the anterior corneal surface. However, if a large air bubble is placed in the anterior chamber so that it contacts the corneal endothelium or if the anterior surface of the cornea is submerged in water, tremendous changes in the refractive power of the eye occur. For example, when the eye is open underwater during swimming, the optical imagery is extremely blurred; the index of refraction of water (1.333) is quite similar to that of the tear film and cornea (1.376). Thus, most of the optical power of the anterior corneal surface is lost. If the air–tear film interface is maintained by the use of a mask or goggles, then underwater vision is as sharp and clear as normal terrestrial vision.

Box 4.1

Keratometry, corneal topography, and corneal tomography

Until recently, total corneal power was usually derived clinically from keratometric or topographical measurements of anterior corneal radius of curvature. The cornea was regarded as a single refractive surface with an effective refractive index of 1.3375, also known as the keratometric index of the cornea, because no accurate measurement of an individual's posterior corneal radius of curvature could be measured clinically. Although not individualized for each patient, this mathematical approximation was used primarily for its convenience in the clinical setting; however, with the advent of clinical Scheimpflug tomography (e.g. Pentacam, Oculus Inc., Lynnwood, WA, USA) and other new tomography instruments soon to come on the market, the problems of accurately measuring an individual's actual posterior corneal surface curvature and corneal thickness along the line of sight in the clinical setting have been predominantly resolved, although finding the exact location of the line of sight is still sometimes cumbersome, particularly in post-refractive surgery patients. Alternatively, total corneal power can still be approximated using these older devices, but at least now more accurately since the keratometric index has been calculated to be around 1.328.

Box 4.2

Contact lenses

The principle of using contact lenses to correct refractive errors is that one essentially replaces the powerful anterior corneal refractive surface to that of the anterior contact lens surface ( Fig. 4.4A , right top diagram). Upon application of a contact lens, the cornea's anterior surface is rendered ineffective as it is bathed with aqueous tears and the air–contact lens interface now becomes the predominate refractive surface of the eye. Soft contact lenses typically are used to treat spherical and regular astigmatism and rarely cause any permanent pathologic changes or alterations to the cornea unless there is a complication, such as corneal infection, infiltrate, or toxic conjunctivitis.

The prevalence of complications in contact lens wearers is currently about 5 percent per year of wear. Soft contact lenses, however, have been noted to cause some acute physiologic changes to the cornea, including epithelial thinning, hypoesthesia, superficial punctate keratitis, epithelial abrasions, stromal edema, and endothelial blebs. They also cause chronic changes including corneal neovascularization, stromal thinning, corneal shape alterations, and endothelial cell polymegathism and pleomorphism (signs of endothelial cell stress). These are all thought to result from the contact lens-induced hypoxia and/or hypercapnia of the tissue.

Most of these physiologic alterations, particularly the chronic ones, are markedly less common now with the introduction of daily-wear high oxygen transmissibility lenses, like silicone hydrogel contact lenses. In contrast, hard contact lenses treat spherical, regular astigmatism, and even some cases of irregular astigmatism. Hard contact lenses cause the same acute and chronic physiologic changes to cornea as soft contact lenses, although they do more commonly cause corneal shape alternations to occur because they induce more mechanical pressure on the anterior corneal surface. In fact, this is the basis for the practice of deliberately fitting tight, overly flat rigid gas-permeable contact lenses with the aim of flattening the central cornea to reduce myopia in a technique known as orthokeratology. The fitting of contact lenses is highly empirical. Considerable trial and error can be involved in adjusting variables such as contact lens material, size, curvature, and other patient-related factors that are used to arrive at an appropriate correction and comfort level for each individual contact lens wearer.

Box 4.3

Refractive surgery

Several keratorefractive surgical procedures have been developed to permanently alter the curvature of the anterior corneal surface, thereby reducing refractive errors. The procedures most commonly performed today use the 193-nm argon fluoride (ArF) excimer laser and include laser in situ keratomileusis (LASIK), a thin-flap variant of LASIK known as sub-Bowman's keratomileusis (SBK) as well as the surface ablation techniques, photorefractive keratectomy (PRK), laser-assisted subepithelial keratectomy (LASEK), and EpiLASIK. The excimer laser reshapes the curvature and contour of the anterior corneal surface by removing anterior corneal stroma in a microscopically precise process known as ablative photodecomposition. This results in non-thermal, photochemical breakage of carbon–carbon covalent molecular bonds in the corneal tissue with submicron accuracy. Thus, excimer laser-based keratorefractive surgery is a very accurate, precise, and safe means to permanently change the curvature and contour of the anterior cornea surface. In fact, it has become the most commonly performed refractive procedure performed in the US since it was approved by the US Food and Drug Administration (FDA) in 1995.

The main reason that photoablation of stroma is effective is that the corneal stroma does not regenerate after it is ablated. It only undergoes reparative stromal scarring that at most replaces 5–20 percent of the ablated stromal tissue. Excimer laser-based keratorefractive surgery has been used to successfully treat myopic and hyperopic refractive errors with mild to moderate degrees of astigmatism resulting in stable long-term (at least 12 years) uncorrected visual outcomes. However, it still is known to potentially deteriorate visual quality due to induction of on- or off-axis aberrations. This occurs mainly because:

(1)
the actual laser ablation profile is sometimes different from the intended profile
(2)
the laser ablation profile is based on spherical geometry whereas the preoperative anterior corneal surface is an aspheric ellipse
(3)
the myopic ablation profile makes the cornea more oblate in shape with a flatter curvature in the center and a steeper one in the periphery
(4)
visually significant subclinical lateral decentrations or torsional misalignments occasionally occur
(5)
the ablation zone is sometimes less than the diameter of the entrance pupil, particularly under low lighting conditions.

The basic principle of myopic correction using the excimer laser is based on the graded removal of central tissue to flatten or increase the radius of curvature of the anterior corneal surface ( Fig. 4.4A , right middle). In contrast, the correction of spherical hyperopia involves the graded removal of peripheral and paracentral tissue to steepen or decrease the radius of curvature of the anterior corneal surface ( Fig. 4.4A , right bottom). Finally, the goal behind various astigmatic ablations is to reshape the anterior corneal surface to bring the two focal points of the eye to the same plane and then ultimately onto the retina with a subsequent second spherical treatment step, if needed. The first step requires selective flattening of the steep meridian and/or steepening of the flat meridian, usually using plus or minus cylinder formats depending on which one removes the least amount of tissue.

Light transmission

The cornea is an excellent example of the structural characteristics that a tissue needs to fulfill its dual role of transparency and mechanical support. Tissue transparency is rarely seen in the animal kingdom outside the eye. In fact, the only structures in humans where this property is seen are in the eye (e.g. cornea, lens, and vitreous). Corneal transparency has occupied scientists for over half a century and initial transparency theories focused on the extracellular stromal matrix while ignoring the cells of the stroma. Corneal transparency is now thought to be attributable to both the lattice-like arrangement of collagen fibrils in the corneal stroma and the transparency of cells that reside in the cornea. In summary, all currently viable transparency theories agree with these major points:

1
Each corneal collagen fibril is an ineffective scatterer of light. Although inefficient, based on the large number of fibrils in the human corneal stroma, destructive interference of scattered light must occur due to the short range order of collagen fibrils in the stroma.
2
Each keratocyte nucleus mildly scatters some light, but since the cell body is an ineffective scatterer of light because of transparent intracellular cytoplasmic water-soluble corneal crystallins, its thinness, and because keratocytes are evenly distributed in the corneal stroma through a clock-wise circular arrangement, light transmission is hardly affected.
3
Scattering of light is minimal in the cornea because it is thin.
4
If an increased refractive index imbalance occurs between fibrils, keratocytes, or extrafibrillar matrix, light scattering can increase tremendously in the corneal stroma resulting in loss of transparency.

In order to understand these theories and generalized principles, one needs to start with the structure of the corneal stroma.

The corneal stroma accounts for 90 percent of the corneal thickness. It is predominantly composed of water (3.5 gram H ₂ O/gram dry weight) that is stabilized by an organized structural network of insoluble and soluble extracellular and cellular substances ( Table 4.1 ). The dry weight of the adult human corneal stroma is made up of collagen, keratocyte constituents, proteoglycans, corneal nerve constituents, glycoproteins, and salts ( Table 4.1 ). Overall, these corneal components work together to maintain and establish a transparent cornea. Although the cornea primarily absorbs most ultraviolet (UV) light, it transmits almost all visible (400–700 nm) and infrared (IR) light up to a wavelength of 2500 nm, with its peak transmission rate of 85–99 percent in the visible spectrum ( Fig. 4.5A ). The remaining portion (1–15 percent) is scattered in all directions by the cornea in a wavelength-dependent fashion with violet light being most affected. Clinical slit-lamp examination and in vivo confocal microscopy suggest that most of the light scatter is due to cellular components in the cornea rather than extracellular matrix. Relative amounts of light scattering due to each stromal constituent are the following: endothelial cells > epithelial cells > nerve cells > keratocytes ≫ collagen fibrils or extracellular matrix ( Fig. 4.5B ). In fact, the in vivo confocal microscope shows that the highest area of the light scatter occurs where differences in the indices of refraction are high, such as at the air–tear film interface of the epithelium. Within the corneal stroma, light scatter predominantly comes from the stroma–plasma membrane interface of nerve cells and the cytoplasm–nuclear interface of keratocytes. With corneal edema or corneal scarring, loss of corneal transparency has been found to be primarily due to changes in the light-scattering characteristics of keratocytes rather than alterations in the extracellular matrix. In these conditions, the cell body of keratocytes scatters considerably more light than normal corneas, particularly their cell bodies and dendritic processes.

Table 4.1

Composition of cornea and sclera

Component	Wet weight (percent)	Dry weight (percent)
Cornea
Water	78	–
Matrix	66
Cellular	12
Collagen	15	71
Proteoglycans	1	9
Keratocytes	1	10
Other	5	10
Sclera
Water	68	–
Collagen	27	77
Elastin	1	2
Proteoglycans	1	3
Fibrocytes	1	3
Other	2	6

Figure 4.5, ( A ) Experimental values for the percentage of light transmitted through normal and edematous rabbit corneas as a function of wavelength. The ratio of the thickness of the edematous corneas to normal thickness values and the number of corneas used for each curve are given in the key.

Collagen

Collagen is a structural protein organized into a relatively inextensible scaffold of water-insoluble fibrils that form the basic structural framework of a connective tissue. The corneal collagens are functionally important in establishing tissue transparency and in resisting tensile loads, ultimately defining the size and shape of the tissue. Collagen molecules measure 1.5 nm in width by 300 nm in length and are composed of a triple helix of three alpha chains. Of the 28 different types of collagen, there currently are 13 known collagen types in the human cornea. Upon secretion from the cell, the propeptide form of the collagen molecule is cleaved and the monomer of the collagen molecule is assembled into fibril-forming, non-fibril-forming, or fibril-associated collagens with interrupted terminals (FACIT) in a surface recess on the keratocyte or, sometimes, begins assembly inside the cell.

The most common collagen molecule in the cornea is type I (58 percent), which usually aggregates into structural, banded fibrils ( Fig. 4.6B ), as seen on transmission electron microscopy, by ordering themselves into a quarter-staggered parallel arrangement that is further stabilized in position by covalent intramolecular and head-to-tail intermolecular immature divalent cross-links in a post-translational enzymatic step using lysyl oxidase ( Fig. 4.6A and 4.6C , top diagram). With increasing maturity, a spontaneous conversion to mature cross-links occurs where intramolecular, intermolecular, and interfibrillar mature trivalent cross-links replace the immature divalent ones resulting in corneal collagen fibrils resulting in more optimal mechanical properties ( Fig. 4.6C , middle diagram). Both immature and mature cross-links occur between lysine and hydroxylysine side-chains. After maturation, the turnover or half-life of collagen molecules and fibrils becomes very slow; the concentration of mature cross-links, however, remains stable, whereas high levels of random intramolecular, intermolecular, and interfibrillar non-enzymatic glycation cross-links accumulate, predominantly between lysine and arginine residues ( Fig. 4.6C , bottom diagram). These non-enzymatic age- or diabetes-related glycation cross-links initially enhance the mechanical properties of fibrils resulting in stiffer, stronger, and tougher fibrils than normal, but occasionally they can go too far, making the tissue too brittle or inextensible to function normally. Type I fibrils are generally heterotypic (type I and type V [15 percent] collagen molecules) since they are composed of two or more types of collagen molecules ( Fig. 4.6B ). This may serve as a fine-tuning mechanism for controlling a fibril's structural characteristics, such as fibril size and interfibrillar connectivity.

Figure 4.6, ( A,B ) Cross-sectional oblique view of a 25 nm diameter, heterotypic, banded (periodicity = 65 nm) corneal stromal collagen fibril composed of type I (white) and V (blue) collagen molecules (bottom). The amino-terminal domains on the type V collagen molecules appear to be important in regulating collagen fibril diameter as they project externally to the fibril surface and presumably block further accretion of collagen molecules through steric and/or electrostatic hindrance effects. The collagen molecules on longitudinal view are aligned in a parallel, quarter-staggered (68 nm) arrangement with 40 nm gaps between molecules (middle). The longitudinal view also clearly shows that the ends of the alpha chains in each collagen molecule form intermolecular cross-links with adjacent collagen molecules as well as intramolecular cross-links (top). ( C ) With maturity, these immature divalent cross-links become mature trivalent cross-links with the addition of interfibrillar cross-link branches. Finally, with aging, intramolecular, intermolecular, and interfibrillar non-enzymatic glycation cross-links form.

Type I fibrils usually reach certain specific diameters based on their composition and ratio of heterotypic collagen molecular types, are restricted from further lateral accretion or growth, and are permitted to fuse and grow axially due to interactions with small leucine-rich proteoglycans covalently bound to its external surface, and through surface interactions connect to various other fibril-forming, non-fibril-forming, or FACIT collagens, which overall links different levels of structural organization ( Fig. 4.6 ). Thus, the surface properties of type I fibrils are a major determinant of intrafibrillar and interfibrillar biomechanical properties of the tissue. The other major determinants are the direction of the fibrils and the suprafibrillar architectures of the tissue, which overall defines the hierarchical structure of the tissue. They typically form uniform 25 nm diameter fibrils in the stroma proper with only slight variability (note: the diameters used in this chapter are based on transmission electron microscopic (TEM); x-ray scattering of ex vivo unprocessed human tissue suggests that a 24–36 percent shrinkage artifact occurs by fixing and processing tissue for TEM studies). Bowman's layer is the main exception as it has uniform 22 nm diameter type I fibrils, which are epithelial in origin as opposed to keratocyte in origin. The diameter of type I fibrils remains constant across most of the central cornea (mean: 25 ± 2 nm; range: 18–32 nm), but then gradually thickens another 4 nm at about 5.5 mm from the center of the cornea, eventually increasing to up to 50 nm at the limbus. Similarly, interfibrillar spacing between nearest neighbor type I fibrils remains constant in the central cornea (mean: 20 ± 5 nm; range: 5–35 nm), then gradually increases another 5 nm at about 4.5 to 5 mm from the center of the cornea before increasing even more rapidly up to the limbus. Type I fibril diameters and interfibrillar spaces do not seem to vary significantly with depth in the cornea. Although the refractive index of type I fibrils (1.47) is different from that of the extrafibrillar matrix (1.35), the highly uniform small size and highly uniform small interfibrillar spaces along with the predominantly parallel directionality of these fibrils results in a highly ordered, lattice-like arrangement. This arrangement is not a true crystalline lattice, but more of a short-range order that allows transparency of the cornea due to destructive interference ( Fig. 4.7 ).

Figure 4.7, ( A ) Low-magnification (×4750) TEM of predominantly orthogonally stacked lamellae in the middle third of stroma proper. ( B ) Higher magnification (×72,500) TEM of two lamellae in the middle third of stroma proper. One lamella is in longitudinal view (top portion) and the other is in cross-sectional view (bottom portion). Notice the uniform 25 nm diameter type I collagen fibrils and uniform 20 nm diameter interfibrillar spaces, which demonstrates only a short-range order, but not a true crystalline lattice arrangement. ( C ) Cross-sectional diagram of collagen fibrils arranged in a true crystalline lattice arrangement. Size of a wavelength of light is shown above for comparison.

Type VI collagen (24 percent) is the second most common type of collagen in the corneal stroma. It is present in an unusually high amount compared to most other connective tissues in the body, but it is also unique in that it is only able to aggregate into repeating tetramers of type VI molecules that are stabilized by disulfide cross-links ( Fig. 4.8A ). Thus, it forms 10–15 nm diameter, non-banded filaments with 20–30 nm diameter beaded ovals having a periodicity of 100 nm. Functionally, type VI filaments act as a bridging structure in the interlamellar space since it binds corneal lamellae together diffusely throughout the stroma where they directly cross one another ( Fig. 4.8B,C ). Along with type XII and XIV FACIT collagens, it also may bridge fibrils together in the interfibrillar space ( Fig. 4.8D ). Overall, this suprafibrillar architecture results in a one-dimensionally-ordered Bowman's layer and a three-dimensionally ordered stroma proper.

Figure 4.8, ( A ) Diagram of a type VI collagen molecule showing how it assembles into filaments by aggregating into repeating tetramers of type VI molecules with a periodicity of 100 nm. ( B ) Low-magnification (×6200) SEM showing bundles of (type VI) collagen filaments extending between lamellae (arrows) and a loose meshwork of collagen filaments on the posterior surface of a corneal lamella where an adjacent lamella crosses it (arrowheads). Bar = 5 µm.

Although difficult to count precisely, the central corneal stroma reportedly consists of approximately 300 corneal lamellae, while the peripheral cornea consists of approximately 500. Although most corneal stromal lamellae extend from limbus to limbus and cross adjacent lamellae at various angles, randomly in the anterior stroma and nearly orthogonal to one another in the posterior two-thirds, various regional differences in lamellar size, directionality, and amount of interweaving are also found ( Figs 4.9 & 4.10 ). The anterior third of the stroma proper has thinner (0.2–1.2 µm thick), narrower (0.5–30 µm wide), and mostly obliquely oriented lamellae (mean 18° ± 11° [range 0–36°]) with extensive vertical and horizontal interweaving ( Fig. 4.9A,B ), while the posterior two-thirds has thicker (1–2.5 µm thick), wider (100–200 µm wide), and mostly parallel-oriented lamellae (mean 1° ± 2° [range 0–5°]) with only slight horizontal interweaving ( Figs 4.9C & 4.10B,C ). In the most superficial layers of the stroma, the interwoven lamellae actually attach or possibly seem to originate from the posterior surface of Bowman's layer in a polygonal fashion creating an anterior corneal mosaic pattern ( Fig. 4.9A , inset) that can be seen on the anterior corneal surface under certain circumstances (the attached fibrils to Bowman's layer seem to be homologous to sutural fibers in the shark cornea and embryologically may be remnants of the primary acellular stroma). Those attaching or originating fibril bundles usually are oriented obliquely to Bowman's layer, but sometimes are noted to be almost perpendicular to it. Finally, except those fibrils and lamellae in the anterior-most region of the stroma that attach to Bowman's layer, the remaining type I fibrils and corneal lamellae stretch across the cornea from limbus to limbus in a belt-like fashion where they turn and form a circumferential annulus approximately 1.0–2.5 mm wide around the cornea. This annulus is thought to maintain the curvature of the cornea, while blending with limbal collagen fibrils.

Figure 4.9, ( A ) The acellular Bowman's layer and anterior-most portion of the stroma proper are shown. The type I fibrils and interweaving lamellae in this region branch extensively and some insert into the posterior surface of the Bowman's layer. This arrangement causes an anterior corneal mosaic pattern to occur on the anterior corneal surface after applying digital pressure through the eyelid and instilling fluorescein. ( B ) The anterior third of the stroma proper has predominantly oblique lamellar orientation and extensive vertical lamellar branching and interweaving. ( C ) The posterior third of the stroma proper along with Descemet's membrane and endothelium is shown. The parallel-oriented, orthogonal arrangement of lamellae in this region of corneal stroma is clearly apparent. Although some collagen fibrils randomly insert into Descemet's membrane, no interweaving or significant attachment occurs in this region of the cornea. BEC = basal epithelial cells. BL = basal lamina. AS = anterior stroma. MS = mid-stroma. PS = posterior stroma. BDM = banded portion of Descemet's membrane. NBDM = non-banded portion of Descemet's membrane. E = endothelial cells. IS = intercellular space of the endothelium. (TEM ×4750).

Figure 4.10, ( A ) The acellular Bowman's layer in cross-section (main photo) and tangential section (inset). Notice the random directionality of the 22 nm diameter collagen fibrils in Bowman's layer. ( B ) The transition zone between the anterior third and mid-third of the stroma proper in cross-section (main photomicrograph) and tangential section (inset). The thin and more obliquely oriented lamellae in this region of the corneal stroma and its lattice-like arrangement of 25 nm diameter type I collagen fibrils are shown. ( C ) The posterior third of the stroma proper in cross-section (main photomicrograph) and tangential section (inset). The thick and parallel-oriented lamellae in this region of the cornea stroma and its lattice-like arrangement of 25 nm diameter collagen fibrils are shown. Compare the orthogonal nature of crossing lamellae (inset) to that in the inset B. (TEM ×72,500).

Keratocytes

Keratocytes make up the second major component of the corneal stroma's dry weight. They are sandwiched between collagenous lamellae forming a closed, highly organized syncytium. They function as modified fibroblasts during neonatal life forming most of the extracellular matrix of the stroma. Subsequently, they remain in the cellular stroma throughout life as modified fibrocytes, where they maintain the extracellular matrix of the corneal stroma. Keratocytes can become metabolically activated or fibroblastic again if the corneal stroma is wounded. The adult human corneal stroma has approximately 2.4 million keratocytes communicating with each other through gap junctions present on their long dendritic processes ( Figs 4.11A & 4.12A,B ). In adulthood, keratocytes occupy 10 percent of the stromal volume, decreasing from 20 percent in infancy, and on two-dimensional, cross-sectional views appear to be sparse, flattened, and quiescent (scant intracytoplasmic organelles) cells lying between corneal lamella ( Fig. 4.11B ). In actuality, keratocytes are three-dimensional, stellate-shaped cells composed of a 15 to 20 µm diameter cell body with numerous dendritic processes that extend up to 50 µm from the cell body. Tangential sections of the normal cornea suggest that these cells more densely populate the stroma than originally thought and are more metabolically active in the resting state than initially presumed since in tangential section an abundance of cytoplasmic organelles are commonly seen ( Fig. 4.11C,D ).

Figure 4.12, ( A ) Reconstruction of keratocyte outlines seen in tangential-section in the anterior and posterior third of the corneal stroma proper.

Tangential sections show that the anterior stromal keratocytes contain twice the number of mitochondria as the posterior two-thirds of the stroma, which correlates with the higher oxygen tension of the anterior stroma. It also has been demonstrated that a higher stromal cell density occurs in the anterior stroma than in the mid- or posterior stroma ( Fig. 4.12A ), whereas a higher cell volume-to-extracellular matrix ratio occurs in the posterior stroma compared to the anterior- or mid-stroma. These views also show that in all levels the keratocytes are highly spatially ordered as they turn in a clock-wise direction like a cork-screw. In vivo confocal microscopy of normal human corneas has shown stromal cell densities averaging around 20,000 keratocytes/mm ³ with a focal zone of increased cell density directly under Bowman's layer, averaging 35,000 keratocytes/mm ³ in the anterior-most layer that gradually tapers to 20,000 keratocytes/mm ³ over the initial 60–100 µm in depth ( Fig. 4.12C ). Confocal microscopy also has shown that stromal cell density decreases with age at a rate of approximately 0.5 percent per year of life with the anterior stroma declining 0.9 percent per year, mid-stroma 0.3 percent per year, and posterior stroma 0.3 percent per year.

Studies using immunohistochemistry or electron microscopy suggest that not all the cells in the corneal stroma are actually keratocytes, but some are one of three types of bone marrow-derived immune cells: “professional” dendritic cells, “non-professional” dendritic cells, and histiocytes ( Fig. 4.13 ). Recent studies also found evidence of a small resident subpopulation of adult stromal stem cells, also known as keratocyte progenitor cells, in the corneal stroma, primarily in the periphery of the corneal stroma near the limbus. The immune cells appear to play a pivotal role in the induction of immune tolerance versus immune initiation in cell-mediated immunity, and the stromal histiocytes have a role in innate immunity as phagocytic effector cells. The presence of adult stromal stem cells helps explain how the slow replacement and renewal of keratocytes occurs after injury, surgery (e.g. epikeratophakia or penetrating keratoplasty [PK]), or toxicity to the central corneal stroma (e.g. mitomycin C).

Figure 4.13, The mouse cornea suggests that 5–10 percent of the cells in the cellular corneal stroma are actually one of three types of bone marrow-derived immune cells. The anterior-third of the cellular corneal stroma contains professional (MHC-positive) dendritic antigen-presenting cells in periphery of the cornea, while non-professional (MHC-negative) dendritic cells are present in both the periphery and center of the anterior cornea. The posterior-most regions of the cellular corneal stroma appear to contain macrophages in the periphery and center of the cornea.

Proteoglycans

Proteoglycans make up the third major component of the corneal stroma's dry weight. They are water-soluble glycoproteins made up of a core protein with a covalently attached anionic polysaccharide side chain called a glycosaminoglycan (GAG). The core proteins are non-covalently attached to collagen fibrils uniformly throughout the tissue, whereas the GAG side-chains extend into the interfibrillar space where it acts as a pressure-exerting polyelectrolyte gel. The cornea collapses to approximately 20 percent of its original volume if the proteoglycans in the corneal stroma are precipitated out with cetylpyridinium. It has become apparent that the primary functions of proteoglycans are to provide tissue volume, maintain spatial order of collagen fibrils, resist compressive forces, and give viscoelastic properties to the tissue as well as having a secondary role in regulating collagen fibril assembly. Corneal proteoglycans previously were referred to as extrafibrillar amorphous ground substance since their water-soluble state made it difficult to fully delineate them with light and electron microscopy ( Fig. 4.14A ). It was not until an electron-dense, cationic dye called cupromeronic blue and a critical electrolyte concentration of 0.1 M MgCl ₂ were used in combination to specifically stain for the sulfate-ester groups on corneal proteoglycans that the shape, size, arrangement, and location of this material were observed with light and electron microscopy ( Fig. 4.14B ).

Figure 4.14, Tangential-section TEM views (×90,000) of longitudinally running type I corneal stromal collagen fibrils in a lamellae without ( A ) and with ( B ) cupromeronic blue staining. Notice the scattered “amorphous ground substance” in the interfibrillar spaces in (A), while in (B) duplexes of proteoglycans are clearly seen bridging next-nearest neighbor collagen fibrils. ( C ) Diagram of how proteoglycans attach along the periphery of type I fibrils via their core proteins (P) and how the core protein tail/GAGs duplex in an anti-parallel fashion in the interfibrillar space between next-nearest neighbor collagen fibrils. ( D ) Diagram of the polymer backbones of keratan sulfate and dermatan sulfate. The top portion shows the primary structure of the repeating disaccharide units of keratan sulfate (top left) and dermatan sulfate (top right). The bottom portion of the diagram shows the secondary structures of each proteoglycan. In the human cornea, ∼50 percent of keratan sulfate is in the normal-sulfated state (left), while the other ∼50 percent is in the over-sulfated state (center). All three displayed proteoglycan polymers have similar backbones and, therefore, form similar secondary structures of a two-fold helix (right). Anionic charges = sulfate esters (X) and carboxylic acid (−).

Since that discovery, it has become apparent that corneal proteoglycans are not amorphous, but rather tadpole-shaped molecules composed of a 10–15 nm diameter globular core protein with a covalently attached 7 nm wide × 45–70 nm in length. The latter of which is where GAG sidechain attaches to. They are arranged in the corneal stroma perpendicular to collagen fibrils with a constant spacing of around 65 nm between each other along the collagen fibrils. Their core proteins non-covalently bind to collagen fibrils in specific gap zones along the peripheral portions of the collagen fibril. The core proteins with dermatan sulfate side-chains bind to ‘d’ and ‘e’ gap zones and those with keratan sulfate side chains bind to ‘a’ and ‘c’ gap zones. GAGs are highly negatively charged, stiff polymers that extend into the interfibrillar space and form antiparallel duplexes with adjacent GAG side-chains ( Fig. 4.14C ), thereby, linking different next-nearest-neighbor collagen fibrils together by forming dumbbell-like structures. The genes that produce the core proteins have been cloned and four types of corneal stromal proteoglycan core proteins have been identified: decorin, lumican, keratocan, and mimecan. Decorin contains a single dermatan sulfate GAG side-chain ( Fig. 4.14D ), while lumican and mimecan have a single keratan sulfate GAG side-chain and keratocan has three keratan sulfate GAG side-chains ( Fig. 4.14D ). Thus, there are four known types of proteoglycan core proteins and only two types of GAGs, keratan sulfate (60 percent) and dermatan sulfate (40 percent), found in the human corneal stroma. GAGs are polymers of repeating disaccharide units of galactose and N-acetylglucosamine or iduronic acid and N-acetylgalactosamine, respectively.

Because the core protein tail and their associated GAG side-chains are post-translationally added to core proteins in the Golgi apparatus, there seems to be some flexibility in how long or how sulfated they can become depending on the function of the connective tissue producing them. The human cornea is unique in that the core protein tail and their associated GAG sidechains are fibril-associated and small in length (KS ∼45 nm and DS ∼70 nm) with a higher amount being over-sulfated than in other connective tissues. A comparative study of corneas from 12 mammalian species suggests that dermatan sulfate is the preferred proteoglycan in oxygen-rich environments, such as in the thin cornea of mice, or is seen predominantly in the anterior portion of the thicker cornea of mammals, such as humans or rabbits. Keratan sulfate is a functional substitute produced through an alternate metabolic pathway in thicker corneas, especially in the posterior portion where oxygen levels may drop precipitously. Functionally, this duality is quite useful because dermatan sulfate appears to be more efficient at holding water; it absorbs less water than keratin sulfate, but holds most of it in a tightly bound, non-freezable state. This is consistent with the fact that dermatan sulfate is more abundant in the anterior corneal stroma in humans, which is the region of highest oxygen tension and most affected by evaporation. In contrast, keratan sulfate is more abundant in the posterior corneal stroma ( Fig. 4.15 ). This is the region of lowest oxygen tension, least affected by evaporation, and the area where the need for loosely bound water is required for transport across the endothelium via the metabolic pumps.

Figure 4.15, ( A ) The regional differences in the corneal stroma for the proportion of the two types of corneal GAGs and the water-absorbing properties in these regions are shown. ( B ) Diagram of the metabolic pathways for dermatan sulfate and keratan sulfate production. The supply of oxygen is the primary factor determining whether dermatan sulfate is made through an aerobic pathway or whether keratan sulfate is made through an anaerobic alternative pathway.

Corneal nerves

The epithelium of the cornea is the most richly innervated tissue of the body with about 16,000 nerve terminals/mm ² (∼2.2 million nerve endings), about 300–400 times more dense than skin. Most of the nerve fibers in the cornea are sensory in origin, responding to mechanical, chemical, and temperature stimuli, and are derived from the ophthalmic branch of the trigeminal nerve (CN III ₁ ) ( Fig. 4.16A ). Refer to Chapter 16 (Sensory innervation of the eye) for full details of the innervation pathway of the cornea. Although all mammalian species have been found to receive variable proportions of nerve fibers in the cornea from the sympathetic and parasympathetic autonomic nervous system, human corneas appear to be on the extreme end of this spectrum as their corneas have a very small proportion of their nerve fibers derived from the autonomic nervous system.

Figure 4.16, ( A ) The nerves of the cornea and sclera are mixed (motor, sensory, and autonomic) and come from the nasociliary branch of the 1st division of the Vth cranial nerve, which branch to form two long (LPCN) and several short posterior ciliary nerves (SPCN). The SPCN supply the posterior sclera, whereas the LPCN supply the cornea and equatorial and anterior sclera. The nerve supply of the cornea is the richest in the body and the sclera also has numerous nerves, commonly with many free nerve terminal endings found in the most vascular portions of the episclera, presumably to regulate blood flow in the anterior segment and to influence aqueous humor outflow.

Electrophysiological studies have shown that the cornea's receptive nerve field primarily is composed of polymodal nociceptors (70 percent) followed by mechano-nociceptors (20 percent) and then cold-sensitive nociceptors (10 percent). Using in vivo laser confocal microscopes, one can only evaluate morphologically the corneal nerves from the main nerve trunks up to sub-basal plexus (SBP) as the resolution and contrast of these images is not capable of visualizing the final nerve branches and free nerve terminals in the corneal epithelium.

Since corneal nerve fibers ultimately terminate in the brainstem, it appears that interneuron intermediate pathways must relay the information to the sensation areas of cerebrum. Additionally, there must also be intermediate relays to efferent systems that trigger the reflex pathways of involuntary blinking via orbicularis motor innervation from CN VII and reflex tearing via parasympathetic innervation of lacrimal gland. The intricate central nervous system (CNS) details of these specific pathways are currently unknown.

Corneal sensitivity is a valuable clinical measure of corneal health since corneal nerves directly maintain the health of corneal epithelium through direct trophic factors as well as serve a protective role in warning the host of possible dangers to the normal healthy state and maintain an adequate basal tear secretion rate. It is usually tested clinically in a semi-quantitative fashion with a Cochet and Bonnet esthesiometer, which is a thin (0.12 mm diameter), flexible, nylon filament of variable length (0–6 cm). When the filament is long, it applies very little pressure to the corneal surface because it bends easily, while when short it applies a proportionally higher pressure before bending. The length is converted into pressure using a conversion table with a range of touch pressures between 11 and 200 mg/mm ² . Corneal sensitivity is defined as the reciprocal of corneal touch threshold and it can be evaluated subjectively by asking the patients when they feel touch upon the cornea or objectively when a reflexive blink response is triggered. Refer to Chapter 16 for full details on the subjective and objective threshold in normal healthy corneas and those with disease or after surgery.

Corneal sensation also variably decreases with corneal disease (e.g. herpes simplex keratitis, diabetes, corneal dystrophies, keratoconus), following surgical procedures on the anterior segment of the eye and sometimes the posterior segment (e.g. panretinal photocoagulation), after application of certain topical medications (e.g. anesthetics, non-steroidal anti-inflammatory drugs), and even after contact lens wear. As nerve regeneration occurs at the rate of approximately 1 mm per month, it may take up to 3–12 months or longer for corneal re-innervation and sensation to maximally recover, depending on the type and degree of surgical injury. After maximal recovery, the sensitivity in the portion of the cornea involved by the procedure often is variably less than that which was present prior to surgery, which means there is a potential long-term as well as short-term neurotrophic keratitis.

It has been recently discovered that after corneal nerve injury, microneuromas can develop during the regeneration process. These microneuromas, as well as injured corneal nerves themselves, exhibit altered functional properties where responsiveness to normal stimuli is impaired, yet paradoxically display abnormal intrinsic electrical excitability (i.e. spontaneous impulses or even abnormal responsiveness to normally minimal stimuli). Overall, this may produce hyperalgesia and/or dysesthesia, which may continue long-term after surgery despite some attenuation. These neuropathic pain impulses are often perceived by the patient as dryness, foreign body sensation, and irritation after surgery, yet are not actually due to actual dryness or irritation of the cornea. These undesired, unpleasant sensations may best respond to ion channel antagonists rather than dry eye lubricating medications.

The mechanisms by which corneal nerves maintain the ocular surface and promote healing after eye injuries are currently under active research in several laboratories. Corneal nerves secrete neuropeptides, such as substance P and calcitonin gene-related protein, and neurotransmitters, such as acetylcholine, vasoactive intestinal polypeptide, and neurotensin, which are believed to be important in corneal epithelial function and proliferation.

Corneal stromal wound healing

The first published report specifically addressing the cellular reactions in the corneal stroma after injury appeared in 1958. It described the morphologic changes of stromal cells after different types of trauma and found that stromal cells lose their interconnecting, dendritic processes immediately after injury with many cells subsequently developing signs of degeneration. That report also described the appearance of morphologically unique, spindle-shaped corneal fibroblastic cells invading into the wound region during later stages of stromal healing. Since that time, many excellent animal model corneal wound healing studies have further addressed the changes in the extracellular matrix and the stromal cells after stromal injury. They suggest that corneal stromal injury is immediately followed by keratocyte apoptosis in the zone around the site of stromal injury with a subsequent influx of transient mixed acute and chronic inflammatory cells, proliferation and migration of surviving keratocytes, and finally differentiation of the keratocytes into transiently metabolically activated cell types called activated keratocytes. This latter cell type is functionally important because it synthesizes and deposits the extracellular matrix of the stromal scar, while also degrading and remodeling the damaged cellular and extracellular tissues around the wound. Epithelial injury alone can also cause transient cellular injury to the underlying stroma presumably from exposure of stroma to tear-related factors, resulting in apoptosis, proliferation, and differentiation into migratory keratocytes as well as resulting in some anterior stromal edema. It, however, does not appear to cause differentiation into activated keratocytes, hypercellularity, differentiation into myofibroblasts, or stimulate extracellular matrix production – all of which are seen with corneal stromal injury whether by incision or excision. Myofibroblasts are characterized by the intracellular cytoplasmic appearance of α-smooth muscle actin, which helps impart contractile properties to the cell.

A number of studies have looked into the expression of cytokines and growth factors in normal and injured corneas. These studies tried to assess the relative importance of each specific factor in cornea wound healing, since it was known clinically and experimentally that epithelial–stromal interactions increase the number of proliferatory and migratory keratocytes within the stromal wound compared to deeper corneal stromal injury, some of which differentiated beyond the activated keratocyte stage into myofibroblastic cell type. Some studies focused even more specifically on strictly epithelial or tear-related cytokines or growth factors since the epithelium and aqueous tears were found to be a major source of cytokines and growth factors. The major cytokines and growth factors studied to date include epithelial growth factor (EGF), fibroblast growth factor (FGF), interleukin-1, nerve growth factor (NGF), transforming growth factor-beta (TGF-β), insulin, retinol, and LPA. TGF-β is currently thought to be the most important growth factor of this group in regard to stimulating a fibrotic reparative stromal scar phenotype. A recent study, however, has shown that local cytokine and growth factor-related influence on normal corneal stromal wound repair is predominantly an early wound healing phenomenon as cell–matrix interactions seem to take over in the later stages of wound healing. For example, integrity or return of a complete epithelial cell basement membrane after stromal injury seems to regulate epithelial–stromal interactions long-term as it decreases the production and, more importantly, the release of TGF-β from epithelial cells into the stroma. One gap in this area of research is how mechanotransduction pathways (mechanical load-induced intracellular signals) fit into this scheme, particularly in maintaining myofibroblast differentiation long-term and thus corneal haze. Other cytokines and growth factors of this group were also found to have some minor complementary or even competing roles with TGF-β, and in other cases they had no role at all in corneal wound healing. For example, NGF is complementary to TGF-β as it too is known to stimulate myofibroblastic cellular transformation independent of TGF-β, but it does so without enhancing keratocyte proliferation or extracellular matrix deposition. In another example, all- trans retinol (vitamin A), which is reflexively secreted in the aqueous tears from its storage location in the lacrimal gland, has a completely different role than wound healing as it maintains the moist, mucosal ocular surface phenotype via gene transcription regulation; thus, it ultimately controls the rate of ocular surface cell proliferation and differentiation (i.e. prevents keratinization and squamous metaplasia). Once activated, keratocytes exhibit a wide range of cellular responses, including increased tritiated thymidine uptake (indicating increased proliferation); initiation of protease and collagenase activity; phagocytosis; interferon, prostaglandin and complement factor 1 production; and fibronectin, collagen, and proteoglycan secretion.

Human studies of stromal wound healing concur with animal studies on most issues with the following notable differences: adult human corneas heal less aggressively, more slowly, and not as completely as animal corneas. However, both animal and human studies show that corneal stromal wounds heal in two distinct phases: (1) an active phase – results in the production of a stromal scar over the first six months after injury in humans, and (2) a remodeling phase – improves corneal transparency and increases wound strength. This 2nd phase occurs up to 3–4 years after injury in humans. Overall, the long-term result in human corneas is the production of a hypercellular fibrotic stromal scar in wound regions where epithelial–stromal interactions occur and a hypocellular primitive stromal scar in wound regions where keratocyte injury pathways occur. These two histological wound types have functional differences as the hypercellular fibrotic stromal scar is strong, but can look clinically hazy because of myofibroblastic cells populating this scar type. In contrast, the hypocellular primitive stromal scar is transparent, but it is very weak in tensile and cohesive strength and serves as a potential space for fluid, inflammatory cells, and microbes. An additional variable to consider in this scheme is the fact that more precisely re-aligned wounds, such as sutured and unsutured wounds with minimal gaping and no epithelial cell plugging, heal better than poorly aligned wounds, such as wounds with wide wound gaping, epithelial plugging, or incarceration of Bowman's layer, Descemet's membrane, or uvea.

Box 4.4

Wound healing variability after common ophthalmic surgeries

The location of scar types along with the variable degree of wound healing responses in the human cornea is best exemplified when one reviews published human histopathology studies that describe findings in corneas that have had common ophthalmic procedures such as cataract extraction (CE), PK, radial keratotomy (RK), astigmatic keratomy (AK), PRK, or LASIK ( Fig. 4.17 ). It is also apparent from these cases that acellular barrier layers, like Bowman's layer, are not reformed if damaged or excised, whereas acellular basement membranes, like the epithelial basement membrane and Descemet's membrane, can be regenerated. Sutured and unsutured clear corneal CE wounds are corneal stromal incisions constructed at oblique angles to the corneal surface so that they self-seal. They usually heal with well-aligned external wound margins and wound edges, which results in a small (50–75 µm in depth) subepithelial zone of hypercellular fibrotic stromal scarring and a remaining deeper zone of hypocellular primitive scarring ( Fig. 4.17A ).

$Figure 4.17, Long-term wound healing findings of corneas that had undergone sutured, temporal, clear-corneal cataract extraction surgery ( A ), astigmatic keratotomy ( B ), photorefractive keratectomy ( C ), and laser in situ keratomileusis ( D ). All corneas shown are greater than 4 years after surgery. (toluidine blue ×15 for A, ×25 for B, and ×100 for C and D).$

Occasional small epithelial plugs are found in the external wound of unsutured clear corneal cataract wounds and sometimes Descemet's membrane is found partially detached or poorly re-aligned along the internal wound margin so that stromal ingrowth occurs. In marked contrast, limbal and scleral tunnel CE incisions heal because fibrovascular granulation tissue from the episclera completely grows into the wound by 15 days after surgery with remodeling up to 2.5 years after surgery (early wound repair mechanisms in vascularized tissue are controlled by bioactive substances released by platelets at the wound site, such as platelet-derived growth factor and TGF-β, as opposed to epithelial-derived factors, such as TGF-β, seen in the avascular cornea). PK wounds heal similarly to sutured clear corneal cataract wounds with the notable differences of having significant wound compression because of the oversized nature of the donor button (usually 0.25–0.5 mm) and wound edge mismatch caused by the irregular, asymmetric nature of the trephine wound found between donor and host wound edges. Additionally, a high percentage of PK cases have overriding external or internal wound edges with Bowman's layer or Descemet's membrane incarceration, which serves to cause weak areas in the hypercellular fibrotic scar or regenerated Descemet's membrane.

Although of partial thickness (70–95 percent depth) and being constructed perpendicular to the corneal surface, RK and AK incisions heal similarly to unsutured clear corneal cataract wounds. The most notable difference from unsutured CE wounds is the more commonly present and more widely variable degree of external wound gaping found in these corneas, which commonly leads to epithelial ingrowth or plugging that rarely goes away long-term ( Fig. 4.17B ). PRK heals entirely under the influence of epithelial–stromal interactions. Therefore, a disk-shaped hypercellular fibrotic stroma scar is produced ( Fig. 4.17C ), which usually is 12–20 percent in thickness of the amount initially ablated. In contrast, LASIK heals similar to that of unsutured clear corneal cataract incisions ( Fig. 4.17D ). A sub-epithelial zone of hypercellular fibrotic scarring occurs at the flap wound margin and the remainder heals by producing a hypocellular primitive stromal scar usually with a thickness around 5–10 percent of the amount initially ablated. However, a notable difference from unsutured CE wounds was that approximately 50 percent of the LASIK corneas were found to have at least some microscopic epithelial plugging present.

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here