Ординатура / Офтальмология / Английские материалы / Corneal Endothelial Transplant (DSAEK, DMEK & DLEK)_John_2010

Active Na+ Transport

Transport of Na+ between tears and stroma has been demonstrated in the rabbit and frog.16-22 In humans,23 when factors such as corneal resting potential and other ion transport systems are taken into account, the net flow of Na+ across the epithelium is from stroma to tears. Nevertheless, the corneal epithelium contains an active Na+ transport system directed from tear to stroma. The transepithelial transport of Na+ is most likely secondary to the Na+/K+ exchange activity of the deeper epithelial membranes, which accounts for the directionality of transepithelial Na+ transport.

Active Cl- Transport

coupling of the chloride secretion to sodium transport. In addition to the transport mechanisms above, the corneal epithelial cells also contain a sodium-hydrogen exchanger and a lactate-hydrogen co-transporter. These transport mechanisms serve to regulate intracellular pH by extrusion of lactate and hydrogen (H+) ions. Corneal epithelial cells have beta-adrenergic receptors that respond to stimulation by activation of adenylate cyclase and increase cyclic adenosine monophosphate (cAMP) levels in cells (Figure 2-2). cAMP increases the conductance of apical chloride channels, stimulating chloride transport. In vitro, the ion transport can osmotically move water from stroma to tears. However, in vivo epithelial ion transport probably has a minor role in corneal deturgescence as compared to the endothelium.29

Cl- transport across the epithelium is from the stroma to tears.19, 24, 25 Net Na+ absorptive transport and net Cl- secretory transport can occur simultaneously only under special experimental conditions. In the living eye, the epithelium generates an electrical potential of about 30 mV, tear side negative, and in this situation the net movement of NaCl across the epithelium appears to be in the stroma- to-tear direction.21 The presence of active Cl- secretory transport in the epithelium raises the possibility that the epithelium might participate in the regulation of stromal hydration in addition to its role as a diffusion barrier. This has been demonstrated in frogs in which Cl- secretion by the epithelium produces significant stromal dehydration.26, 27

Electrophysiology of Corneal Epithelium and Ion Transport

The mammalian cornea generates a transepithelial potential of 25-35 millivolts. This high voltage is consistent with the low ionic conductance of the optical epithelial cell membranes and high resistance of the tight junctions of the paracellular pathway. About 50% of the short circuit current across the corneal epithelium is carried by chloride ions moving through the apical membrane channels into the tears. This current is due to ionic gradients set up by epithelial transport of sodium and chloride ions. Ouabain sensitive Na+ - K+ ATPase present in the basolateral membrane of these cells, pumps sodium ions from the cells towards the stroma.28

A sodium-chloride co-transporter, also located in the basolateral membrane, facilitates the influx of sodium down its electrochemical gradient carrying with it chloride ions, which then diffuse through channels in the apical membrane. This chloride secretion is blocked when the Na+ - K+ ATPase is inhibited by Ouabain, demonstrating the

Figure 2-2: Scheme for the neuroregulation of Cl¯ transport in the corneal epithelium. It is proposed that serotonin and dopamine can evoke the release of norepinephrine from the sympathetic nerve fibers in the cornea. In turn, norepinephrine may activate adenocyclase via the β-adrenoreceptor to increase cell levels of cyclic AMP and, finally, to increase the chloride conductance of the apical epithelial membrane.

Physiology of the Corneal Stroma

The corneal stroma is basically an extracellular compartment with keratocytes and nerves. It measures around 470 micrometers thick centrally in a human adult cornea. Collagen fibers approximately 22 to 32 nm30,31 in diameter appear to run uninterrupted from limbus to limbus in flat sheaths or lamellae. Although the concentration of Na+ and K+ may collectively be 35 mEq/L higher in the stroma than in the aqueous humor,32 the combined activity of these ions, and hence their effective osmolarity, is probably less in the stroma than in the aqueous humor. This fact is important for control of corneal hydration. When

the stroma swells, the diameter of collagen fibrils remains constant; swelling takes place in the ground substance, which is rich in glycosaminoglycans, and leads to an increased spatial separation of collagen fibrils.33

The stroma is maintained in a relatively dehydrated state, in comparison to its ability to swell. The stroma consists of 78% water, which is equivalent to a ratio of 3.45 parts water (by weight) to 1 part solid material. The corneal stroma scatters less than 10% of normal incident light. This is an unexpected property of the cornea given the disparity in refractive index between the collagen fibrils and the proteoglycan matrix. Maurice34 proposed that corneal transparency is a consequence of a crystalline lattice arrangement of collagen fibrils within the stroma. He also proposed that the light scattered by individual fibrils and uniform diameter is canceled by destructive interference with scattered light from adjacent fibers; therefore light is scattered only in the forward direction. Such an arrangement requires that all collagen fibrils be of equal diameter (275-350A) and that all fibrils be equidistant from each other. However, to maintain this transparency, it is required that the distance between the collagen fibrils be less than one half the wavelength of visible light. On the other hand, Goldman and Benedek35 and others36, 37 recognized that refractive elements in tissues whose dimensions are small (<200 nm) compared with the wavelength of light should not scatter as much as light as might be predicted by the stringent requirements of the crystalline lattice theory. Light scattering is wavelength dependent. This dependence of corneal transparency on the distribution and size of collagen fibrils is supported by observations of swollen corneas and by the structure of opaque sclera. When the epithelial or endothelial barrier of the cornea is damaged, the stroma imbibes water and swells, leading to loss of corneal transparency. This uptake of water causes formation of “lakes” devoid of collagen fibers within the stroma. This causes increased divergence of refractive index within the stroma as well as an increase in distance between collagen fibrils leading to a wavelength – dependent loss of light transmittance that increases with the amount of corneal swelling. It was found that fibril diameter is greater in the anterior cornea than in the posterior cornea and that the density of fibrils is lower in the anterior cornea than in the posterior cornea in both rabbits and humans. This leads to two-fold (in humans) and three-fold (in rabbits) increase in light scattered by the anterior cornea as compared with the posterior cornea. Proteoglycans are responsible for maintaining the regular spacing and packing of the collagen fibrils which is the basis of corneal transparency. The presence of functional epithelial and endothelial barriers and a metabolic pump

to maintain the corneal water content at 78% normally maintain the biochemical and physical properties of the corneal stroma and ultimately corneal transparency is maintained.34

Physiology of Corneal

Endothelium

The corneal endothelium is composed of a single layer of hexagonal cells which forms the posterior corneal surface. It is approximately 400,000 cells and 4-6 micrometers thick. The posterior cell membrane is thought by some to be coated with a viscous substance,38, 39 possibly of endothelial origin, which may reduce lipid membrane surface tension to promote wetting. A single primary cilium has been demonstrated in many endothelial cells, but its function is unclear.40, 41 Endothelial cells lack the ability to undergo mitosis. However, they do have the ability to enlarge and to maintain tight apposition with neighboring cells, preventing excessive diffusion of the aqueous into the stroma. The endothelium acts as a permeability barrier that restricts the movement of water and solutes into hydrophilic stroma. If the integrity of this layer is broken, corneal edema rapidly develops.

Normally, the endothelium enjoys a privileged and protected environment in the anterior chamber, but it remains a fragile cell layer whose integrity and viability must be guarded to ensure the success of any intraocular procedure.42 Endothelial cell density is an important parameter to assess the condition of the cornea. The specular microscope has been a useful tool for noninvasive valuation of endothelial cell densities (Figure 2-3).43 Confocal microscopy is considered another important tool

Figure 2-3: (A) Specular photomicrograph of the corneal endothelium in a 28-year-old patient. The cell density is approximately 2,800 cell/ mm2. The dark bands are artifacts from applanation. (B) Endothelial cell density of approximately 400 cells/mm2 in a clinically successful corneal graft. Note the large cell size, pleomorphism and polymegathism.

to assess the integrity of the endothelium. Cell density varies with age. At birth, cell densities range from 35004000 cells/mm2, whereas the adult corneal normally has densities of 1400-2500 cells/mm2. Corneal transplants may have fewer than 1000 cells/mm2 and remain clear. A lower limit to this ability occurs at densities of 400-700 cells/ mm2, below which corneal edema and loss of vision ensue.

When endothelial cells are subjected to stress, and especially when some cells are lost, the remaining cells may lose their regular hexagonal shape and become irregular in shape (pleomorphism) and size (polymegathism). These changes can occur with age, after trauma, and in long-term contact lens wear. The significance of these changes is unclear, but there is evidence that a cornea with these changes cannot withstand additional trauma as well as a normal cornea.

Endothelial Barrier and Pump Functions

The corneal endothelium is leaky compared to the epithelium. In a physiologically normal and transparent cornea, aqueous humor crosses the endothelium and enters the stroma at a slow but constant rate. This constant leak of aqueous provides the principal source of glucose, amino acids and other nutrients for the cells of the avascular cornea. The permeability of the endothelial barrier results from the presence of low resistance intercellular junctions at the cells’ apical membrane. This leaky property made classic tracer experiments to demonstrate net ion transport mechanisms44 technically difficult. Nevertheless, the endothelium is clearly demonstrated to transport bicarbonate from the stroma to the aqueous humor in amounts sufficient to explain the simultaneous isotonic transport of fluid.45, 46 Subsequently, the transport of sodium ions in the same direction was inferred.47 Although the specific details of these transport mechanisms are unclear, several different schemes appear in the literature,48-50 indicating involvement of several alternate anion pathways.51 Since fluid and solutes are continuously entering the stroma, the maintenance of corneal thickness and transparency is dependent on the active removal of fluid that leaks into the stroma. A constant corneal thickness is maintained when the volume of fluid leaking into the stroma is equal to the volume of fluid actively removed from the stroma by the endothelium through the endothelial pump. Transport enzymes and ion channels in the endothelial cell membrane transport ions in a stroma-to- aqueous direction.52 The osmotic gradient generated by this active transport mechanism draw water out of the stroma into the aqueous humor. The effect of endothelial ion transport results in net fluxes of sodium and bicarbonate

ions from stroma to aqueous. Fischbarg and coworkers53 promote the idea that endothelial fluid transport involves electroosmosis through the intercellular junctions as the primary process in a sequence of events secondary to active ion transport. Ruberti and Klyce54 report that transendothelial fluid transport may be rapidly self-modulating to control stromal hydration in response to small osmotic stresses, and this may assist in the regulation of corneal hydration.

Corneal endothelial carbonic anhydrase, which catalyzes the conversion of carbon dioxide (CO2) and water (H2O) into bicarbonate (HCO3) and hydrogen (H+), is believed to provide an important source of bicarbonate for the endothelial pump. Carbonic anhydrase inhibitors have been shown to inhibit both corneal deturgescence and the electric potential across the endothelium. Na+ - K+ ATPase is an integral membrane protein localized to the lateral cell membrane in corneal endothelium. Ouabain, a specific inhibitor of this enzyme, has been shown to prevent temperature reversal of enucleated eyes and causes corneal swelling as it inhibits Na+ - K+ ATPase at doses comparable to those that cause corneal swelling.

The endothelial cells in the human cornea decline constantly through out life. Despite this constant loss of cells, normal thickness and transparency are maintained. Other factors imposed on the endothelium markedly augment the normal aging process accelerating endothelial cell loss as in intraocular surgery, dystrophies, degenerations, glaucoma, and drug toxicity. When cell density declines to several hundred cells per square millimeter, corneal decompensation occurs. Corneal endothelium possesses a large reserve capacity in terms of the density of cells required for the maintenance of corneal transparency. If endothelial damage results in corneal edema, the restoration of the normal corneal hydration will be dependent on the extent to which the balance between barrier and pump functions can be reestablished.55

Corneal Metabolism and Nutrition

Corneal metabolism is dependent on oxygen derived mainly from the atmosphere, the aqueous and the limbal vessels (Figure 2-4). The normal O2 in aqueous is low (40 mm Hg) compared to the tears (155 mm Hg). Most of the metabolic requirements for glucose, amino acids and vitamins are supplied through the aqueous humor and, to a lesser extent, via the tears and limbal vessels. Under both aerobic and anaerobic conditions glucose is diverted to the hexose monophasphate shunt (HMP) regulating levels of (NADPH) and converting hexoses to pentoses utilized in nucleic acid synthesis. Glucose derived from the aqueous

Figure 2-4: Conceptual scheme of oxygen supply to the cornea. Although some oxygen enters the cornea from the limbus and aqueous humor, the anterior cornea derives essential amounts from the tears.

or from epithelial glycogen stores is converted to pyruvate by glycolysis under anaerobic condition yielding two molecules of ATP (adenosine triphosphate). Under aerobic conditions, pyruvate is oxidized in the Krebs or tricarboxylic acid cycle to yield water, carbon dioxide and 36 molecules of ATP per cycle. Under hypoxic condition as during contact lens use increasing amounts of pyruvate are converted by lactate dehydrogenase to lactate which diffuses from the epithelium into the stroma leading to epithelial and stromal edema.52

Corneal Avascularity

The human cornea is normally avascular and transparent. Corneal neovascularization results from a variety of diverse conditions. The avascularity of the cornea is due to the compactness of the stromal cells, which acts as a mechanical obstacle. Also, the stroma is rich in mucopolysaccharides which is a barrier to neovascularization. Causes of corneal neovascularization include ocular surface diseases such as acne rosacea and staphylococcal hypersensitivity, immunological disorders such as cicatricial pemphigoid, corneal allograft rejection and collagen vascular disease with peripheral corneal ulceration and neovascularization. Corneal neovascularization is also found in keratitis caused by herpes zoster, a result of sprouting from perilimbal vessels. New capillaries arise from the perilimbal capillaries by focal degradation

of venular basement membrane followed by movement of the endothelial cells towards the stimulus. Endothelial cell proliferation occurs to form a solid sprout, which later develops a lumen. Two sprouts join each other to form a loop, the outer surface of which is lined by pericytes and blood flow begins. The process is repeated again by sprouting from apex of the loop. The angiogenic process consists of three basic steps:

1.Enzymatic degradation of basement membrane.

2.Endothelial cell movement.

3.Endothelial cell proliferation.

The specific angiogenic factors include acidic fibroblast growth factor (FGF), basic FGF and transforming growth factor.56

Corneal Innervation

The cornea is richly supplied with sensory nerves which are derived from the ciliary nerves originating from the ophthalmic division of trigeminal nerve. The cornea is surrounded by a perilimbal nerve ring from which fibers penetrate the middle and anterior layers of the stroma extending radially towards center of the cornea. The nerve fibrils divide dichotomously emerging from the deeper layers of cornea penetrating Bowman’s membrane to a subepithelial plexus. From the plexus, axon terminals spread among the epithelial cells innervating all layers of epithelium with sensory receptors. Adrenergic sympathetic nerves that originate in the superior ganglion also innervate the cornea. So it is one of the most sensitive tissues of the body which serves as a protective function. It contains A delta and C fibers, which respond to mechanical, thermal and chemical stimuli. They usually have the lowest threshold for mechanical stimulation. Neuropeptides including substance P and calcitonin gene-related peptide (CGRP) are present in corneal nerves and appear to have a direct but poorly understood trophic effect on the epithelium.28

Control of Corneal Hydration

Several hypotheses have been proposed to explain the control of corneal hydration. Several components of corneal physiology are involved in this process (Figure 2-5). Underlying each of the hypotheses is the fact that stromal imbibition pressure (IP) must be counteracted to prevent swelling. In the past, it was suggested that although the cornea is surrounded by fluid, it would not swell if the cell membranes were impermeable to either water57 or salt.58, 59 Although both the epithelial and endothelial cell membranes have defined barrier properties, neither

Figure 2-5: Factors and forces involved in the control of corneal stromal hydration. Intraocular pressure and stromal imbibition pressure are forces that promote water accumulation in the stroma. Ion transport pumps in the corneal membranes reduce the osmotic pressure of the stroma such that the semipermeable membrane properties of the epithelium and endothelium balance the forces promoting edema.

membrane is impermeable to water or electrolytes.60 The effect of evaporation from the tear film has also been suggested as a major source for the control of corneal hydration61 and, in fact, the cornea is 5% thinner during waking hours than during sleep.62, 63 Disruption of the lipid layer of the tear film leads to evaporation from the epithelial surface, which can lead to dellen formation. High postoperative intraocular pressure can promote more rapid clearing of a corneal graft by accelerating the movement of fluid through the anterior corneal surface, thereby thinning the cornea. However, elevated intraocular pressure can also cause epithelial edema, as well as stromal swelling, in certain refractive surgical procedures.64

Maurice60 first proposed that, because they are not impermeable to solute or water, the corneal membranes could prevent corneal edema if ions were actively transported out of the stroma as fast as they leaked in by passive means (solvent drag, diffusion). Such a process would, in essence, lead to the sustaining of an osmotic gradient (2 to 3 mOsm) between the corneal stroma and the external solutions, which would balance the swelling pressure of the stroma. Davson65 and Harris and Nordquist66 demonstrated that corneal hydration is loosely linked to the metabolic activity of corneal membranes.

Corneal Edema

Corneal edema can be acute or chronic. Although acute corneal edema, as can be seen in contact lens wear and in angle-closure glaucoma, is often reversible, chronic corneal

edema is usually irreversible and treatment varies depending on the nature of the underlying problem. Chronic corneal edema develops as a consequence of endothelial dysfunction, regardless of whether the original problem was dystrophy, inflammation or trauma.

Endothelial dysfunction can be in the form of increased permeability, decreased ion transport function or both. In mild cases, increased stromal thickness occurs initially with minimal vision affection. In advanced cases, epithelial edema ensues, which affects vision dramatically. In late stages, painful bullous changes can develop. Thick subepithelial pannus eventually develops, leading to disappearance of the bullae.

Epithelial Edema

Epithelial edema resulting from endothelial dysfunction or elevated intraocular pressure is predominantly extracellular.67 The underlying pathophysiologic mechanism seems to involve a forward movement of stromal fluid and aqueous, generated by the IOP. Thus if the endothelial functional reserve falls below a certain level, leading to edema and a reduction in stromal swelling pressure to below the value of the IOP, fluid from aqueous humor can collect.68 Because the otherwise healthy epithelium has such a high resistance to electrolytes and to the flow of water, the fluid can be trapped within the epithelium, resulting in the formation of cysts and bullae. The concept that the IOP is the driving force for the fluid movement is particularly supported by the fact that in phthisis bulbi with marked hypotony, epithelial edema does not occur, no matter how damaged the endothelium is and how thick the stroma is.42

Stromal Edema

When the endothelial cell density falls below a critical level (200-400 cells/mm2), the ability of the endothelium to maintain stromal hydration begins to falter and the edema develops gradually.42 Because the stroma can swell only in the posterior direction, its thickness increases especially centrally. This flattening of the posterior surface can throw Descemet’s membrane into multiple folds.

Endothelial Changes

Under stress, e.g. trauma, the endothelium changes in a way characterized by decreased cell number and enlargement and irregularity of the shape (polymegathism and pleomorphism).69 In chronic inflammation, the endothelium may undergo fibrous metaplasia.70 In Fuchs’ dystrophy the cells exhibit a change in form and show

vacuoles, phagocytized pigment, and irregular depositions (guttata). Even in advanced cases of Fuchs’ dystrophy, the endothelial surface appears intact.71, 72

Acknowledgements

All figures were taken with permission from Kaufman HE, Barron BA, McDonald MB, Waltman SR, eds. The Cornea. Boston: Butterworth-Heinemann, 1988: 3-54.

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Introduction

The corneal endothelium separates the anterior chamber from the subjacent stroma. This unique monolayer is primarily responsible for control of corneal hydration (See also, Chapter 2, Corneal Physiology). It is essentially incapable of mitosis and, if damaged, causes corneal edema and loss of vision.

Embryology and Development

Corneal development is induced by the separation of the lens vesicle from the surface ectoderm at around 33 days of gestation. The basal epithelial cells of the surface ectoderm secrete collagen fibrils and glycosaminoglycans that form the primary stroma. At five weeks gestation, the first of three waves of neural crest cells migrate along the posterior surface of the primary stroma to form the initially multilayered corneal endothelium.1-3 By the third month of gestation, the double-layered endothelium thins to a monolayer of endothelial cells that begin to deposit Descemet’s membrane. Descemet’s membrane initially consists of the anterior lamina densa and the posterior lamina lucida, which is adjacent to the endothelium. Between the third and fourth month of gestation, gap junctions and the apical band develop. Descemet’s membrane then goes on to form a complete layer of the fetal banded zone, which reaches a maximum thickness of 3 μm at birth. The endothelium shares its neural crest origin with stromal keratocytes, the sclera, the cells of the anterior iris, and the trabecular meshwork.4 This may explain the observation that congenital diseases of the endothelium often involve related structures in the anterior segment of the eye. Further evidence of this relationship is suggested by studies of genetic mutations affecting neural crest cell differentiation, providing insight into such conditions as Axenfeld-Rieger syndrome and Peters anomaly.5 The normal human fetal cornea appears to be primarily devoid of lymphatic and blood vessels and remains avascular into adulthood,6 an important factor contributing to the relative immune privilege of the cornea after penetrating keratoplasty and other procedures.

Morphological Characteristics,

Aging and Wound Repair

The corneal endothelium is composed of a monolayer of mostly hexagonal cells, covering the posterior surface of the Descemet’s membrane. Human corneal endothelial cells (HCECs) are 5 μm thick and 20 μm wide, with a surface

area of 250 μm2. In addition, they have numerous interdigitating cellular processes that increase the area of contact.7,8 In the early prenatal period, there is a rapid increase in total HCEC number through mitosis, while later, enlarging endothelial cells cover the rapidly growing surface of the cornea without a significant change in cell density.9 The HCEC density is highest at birth with about 500,000 cells and an average of about 4000 cells/mm2, although numbers as high as 7500 cells/mm2 have been reported.10,11

The endothelial cell density (ECD) decreases throughout life at variable rates. From birth to 14 years the rate of endothelial cell loss is approximately 3% per year. After age 14, endothelial cell loss slows to about 0.6% per year. Specular microscopy in normal young adult corneas reveals an ECD of about 3500 cells/mm2.12 The ECD declines to about 2000 cells/mm2 in older age.13 As ECD decreases, individual cells enlarge and lose their hexagonal shape.2,14 The critical ECD below which the cornea decompensates is approximately 300-500 cells/mm2. 15 (See also Chapter 2, Corneal Physiology). The coefficient of variation of mean cell area (CV) is about 0.25 in the normal cornea, and is a clinically valuable marker. An increase of the CV is termed polymegathism which normally occurs with increasing age, or when the endothelium is stressed or traumatized. Another morphometric parameter is the hexagonality of the endothelial cells, which is 70-80% in the normal cornea. Deviation from hexagonality is termed pleomorphism, which is also seen with aging or in various endothelial disease states. Factors such as gender and ethnicity have also been noted to affect endothelial cell morphology and density in healthy subjects.16,17 There are important differences between the peripheral and central HCECs. The ECD is higher in the periphery as opposed to the central cornea. In addition, a higher replicative competence has been demonstrated in peripheral HCECs versus central HCECs.18 The endothelium forms the anterior border of the anterior chamber, and is therefore susceptible to blunt or penetrating trauma such as cataract extraction or anterior chamber intraocular lens implantation, as well as inflammatory and other conditions (Figures 3-1 to 3-4). As Edelhauser discussed in his 2006 Proctor Lecture, the peripheral HCEC population near Schwalbe’s line may represent an endothelial stem cell reservoir.19 With further investigation, findings such as this will improve our understanding, and avoidance, of critical areas of the endothelium during intraocular surgery.

Recent studies revealed some factors that contribute to the observable age-related changes of HCECs. From in vitro and graft survival studies, the survival of endothelial cells