Corneal Endothelium Lecture 2 PDF

Corneal Endothelium Roy Joseph PhD MBA School of Optometry and Vision Sciences August 26th 2024 Schematic representation of the eye Corneal epithelium 50 m,10% of total Five layers of cornea...

Corneal Endothelium Roy Joseph PhD MBA School of Optometry and Vision Sciences August 26th 2024 Schematic representation of the eye Corneal epithelium 50 m,10% of total Five layers of cornea Bowman’s layer, 8-4 m Stroma, 500 m, 90% of total Descemet’s membrane, 10 m Endothelium, 5 m  Lamellae Str o ma Descemet’s membrane Endothelium Hexagonal shape Endothelial mosaic Examples of Primary Endotheliopathies Corneal guttata Fuchs endothelial corneal dystrophy Posterior polymorphous endothelial dystrophy Congenital hereditary endothelial dystrophy Iridocorneal endothelial syndrome Senile endothelial degeneration  Endothelial Cell Function Transport of nutrients from Maintenance of aqueous into cornea cornea deturgescence Oxygen Glucose amino acids (maintains (mainly from and vitamins corneal tear film) Nutritional transparency) transport Paracellular Endothelium is leaky Factors for correct route Na+/K+-ATPase hydration: Apical surface retards Tight zonular junctions bulk flow from aqueous Endothelial tight to stroma junctions Pump-leak hypothesis Na+/K+-ATPas (In epithelium and endothelium)  Corneal Endothelium Pump-leak hypothesis Primary suggests that an Endothelium also function: (1) To equilibrium is secrete an maintain needed in the anteriorly located corneal amount of passive basement transparency fluid flow into the membrane called by regulating cornea and energy Descemet’s corneal expended pumping membrane and a hydration and out excess fluid for posteriorly located (2) nutrition maintenance of glycocalyx layer. supply through corneal a leaky barrier transparency and and a relative dehydration metabolic of corneal stroma. pump function.  Pump leak hypothesis Two major functions of the endothelium are: 1. The ability to permit transport of (Collagen, GAG and nutrients from proteoglycans) aqueous humor Na+,K+ into cornea. ATPase Nutrients 2. Maintenance of cornea in the Leaky relative state of tight Aqueous Water junctions deturgescence needed to ensure corneal transparency.  IS = intercellular space (20 nm) 65-year-old patient with healthy corneas Poerior corneal stroma, Descemet's Tight membrane, and corneal endothelium Junctions from a 65-year-old patient PS = posterior stroma. BDM = banded Nucleus portion of Descemet's membrane. NBDM = non-banded portion of Descemet's membrane. E = endothelial cells Figure 4.22 (A) Scanning electron micrograph (×1000) of the posterior surface of the corneal endothelium from a 65-year-old patient with healthy corneas. Note how the hexagonal endothelial cells form a uniform monolayer with small 20 nm intercellular spaces between adjacent endothelial cells. E = endothelial cells. IS = intercellular space. (B) Transmission electron micrograph (×4750) of the posterior corneal stroma, Descemet's membrane, and corneal endothelium from a 65-year-old patient with healthy corneas. PS = posterior stroma. BDM = banded portion of Descemet's membrane. NBDM = non-banded portion of Descemet's membrane. E = endothelial cells. IS = intercellular space. (C) Immunofluorescent laser confocal microscopic photomicrograph (×2000) of human corneal endothelial macula occludens tight junctional complexes stained with immunolabeled monoclonal antibodies to junctional adhesion molecule-A (green). Nuclei are counterstained with TO-PRO (blue). (Courtesy of Kenneth J. Mandell, MD, PhD.) Specular Microscopy  What specular microscopy reveals about the patient's endothelium Evaluate the corneal endothelium and be able to differentiate between normal endothelial structure vs. abnormal endothelial structure. Identify the symptoms of moderate to advanced endothelial disease, such as blurred vision, fluctuating vision, or permanent visual impairment. Explain the importance of endothelial cell density, and the reasons why it may change. Recognize the characteristics of different corneal endotheliopathies using specular microscopy, including corneal guttata, Fuch’s endothelial dystrophy, age-related endotheliopathy, contact lens-induced endotheliopathy, and others.  6X increase Entire posterior corneal surface without gaps Normal permeability of the human endothelial cell monolayer to carboxyfluorescein. Figure 4.23 (A) The normal barrier function of corneal endothelium is due to endothelial cells covering the entire posterior corneal surface without gaps and the focal, discontinuous tight junctions in its apical intercellular space. (B) shows the normal permeability of the human endothelial cell monolayer to carboxyfluorescein (2.26 × 10−4 cm/min) compared to that without endothelium (12.85 × 10−4 cm/min), which resulted in a six-fold increase in permeability. (Modified from Watsky MA et al. Exp Eye Res 1989; 49:751–67.)  Corneal Endothelium Morphology and Ultrastructure Single cell layer (5 m) with Polymegathism: apical surface facing anterior Uniformity of cell size chamber and basal surface to Pleomorphism: Decrease the Descemet’s membrane in hexagons Hexagonal (70-80%of all the Cell density: cells) -At birth: 4000 cells/mm2, --- Leaky tight junctions at cell At 8-9th decade: 1000-2000 apex cells/mm2 Cell numbers: At birth: total Minimum cells required for cells: 400,000, Density: 3500- transplant: 400-700 4000/mm2,Thickness 4-6 m cells/mm2 and Cell width: 20 m  Endothelial Morphometry Determination: Measure the area of apical membranes of a population of cells and calculate the coefficient of variance (CV) of cell size. Normal CV is about 0.25. An increase means variable cell size and cells are in distress. Polymegathism : A decrease in hexagons (size) with concomitant increase in number of cells with more or fewer than six sides. Cell size (Polymegathism) can vary significantly with no change in cell density  Cell Density of Corneal Endothelium Decreases with Age At birth: 4000 cells/mm2 At 8th or 9th decade: 1000-2000 cells/mm2 Minimum number of cells needed for endothelial function: 400-700 cells/mm2  Effect of penetrating keratoplasty (full thickness corneal grafting) on endothelial cell density Cell density at graft: 1000 cells/mm2 Cell loss continues for 6 months and then cornea stabilizes Remodeling of cornea continues for 2 years Normal endothelium Graft endothelium  Effect of cataract surgery Effect of contact lens wear on on endothelial cells endothelial cells Pleomorphism: 46 year-old non-contact lens wearer Percent cell loss 46 year-old wore hard contact lenses for 27 years. Polymegathism Pleomorphism: Decrease in hexagons; Polymegathism: Uniformity of cell size  Corneal diameter Nuclei/100 m length  Inset showing data using a uniform scale Nclei/100 m length  Age-related Cell loss from human corneal endothelium Increase in cell loss Limbal Paracentral Central (B) Graph illustrating the central, paracentral, and peripheral corneal ECDs for healthy, normal subjects of different ages. (From Edelhauser HF. Cornea 2000; 19:263–73; and Edelhauser HF. Invest Ophthalmol Vis Sci 2006; 47:1755–67.)  Pump-Leak Hypothesis 1. Leaky barrier function (Tight junctions) 2. Metabolic pump function (Na,K- ATPase) Two major functions of the endothelium are: 1. The ability to permit transport of nutrients from Na+,K+ aqueous humor ATPase Nutrients into cornea. 2. Maintenance of Leaky cornea in the tight Aqueous Water junctions relative state of deturgescence needed to ensure corneal transparency.  Leaky Barrier (Tight Junctions) Function of Endothelium Macula occludens tight When endothelial cells Barrier function is junctions: Characterized by: are disrupted, the dependent on: 1. Partial obliteration of corneal permeability 1. Sufficient intercellular space and a increases 6X. When number of cells. partial retention of 10 central ECD decreases 2. Intact macula nm wide intercellular below 2000 cells/mm2 the occludens (tight space. compensatory metabolic junctions) 2. In healthy corneas: pump mechanism keeps between The barrier prevents bulk the cornea at normal endothelial cells flow of fluid from dehydrated state until resulting a low aqueous humor to the central ECD of 500 electrical corneal stroma, but cells/mm2 resistance to allows moderate aqueous humor diffusion of small flow. nutrient, water and other metabolites to cross into stroma through 10 nm wide intercellular space.  Pump-Leak Hypothesis Rate of leakage of water and solutes into corneal stroma from aqueous Balanced (via ion pumping by Na+,K+ATPase) Rate of pumping of excess of water from the stroma back to aqueous Imbalance Corneal swelling Na+,K+ATPase Operates at constant Rate of leakage proportional to rate pressure gradient across the endothelium (Self-adjusted homeostasis mechanism)  When the leak rate equals the metabolic pump rate, the corneal stroma is 78 percent hydrated and the corneal thickness and transparency is maintained. (C) The opposing forces of the leaky corneal endothelial barrier and the metabolic pump sites are shown. When the leak rate equals the metabolic pump rate, the corneal stroma is 78 percent hydrated and the corneal thickness and transparency is maintained.  Metabolic Pump Function Transporters located at The Na+/K+ ATPase numbers are basolateral membrane, quantified by using [+H]-oubain, which and transport ions (Na+ binds at 1:1 ratio with pump. and HCO3-) out of 3X106 pump sites/single endothelial stroma to aqueous. cells. This corresponds to 4.4 trillion sites /mm2 of an intact endothelial cells. An osmotic gradient is Factors that alter endothelial pump function: created and water is 1. Pharmacological inhibition of Na+/K+ ATPase, osmotically drawn from 2. Decreased temperature. stroma into aqueous 3. Lack of HCO3-, carbonic anhydrase inhibitors. humor 4. Chronic reduction of ECD from mechanical injury or disease state Osmotic gradient Compensatory mechanism can be maintained At ECD between 2000 and 750 only if endothelial cells/mm2: Increase in the activity of At ECD barrier is maintained. pump sites with more ATP production 750cells/mm2: and/or increasing the total number Compensatory and density of pump sites on lateral mechanism fails membrane.  Oubain A crystalline glycoside derived from the seeds of Strophanthius gratus and the wood of Acocanthera oubaio. It blocks Na+,K+-ATPase activity and often used for pharmacological studies because of its greater solubility in water. It is also used as an arrow poison in Africa.  Retardation of Bulk Flow of water from aqueous through Endothelium Resistance to bulk flow Gap junctions Tight junctions (Secondary, act as selective (primary) filters) Normal: 25-40 nm Corneal endothelium: 3 nm 10-20 nm intercellular space Gap junctions act as selective barrier  Retardation of bulk flow via endothelium (continued) Molecular Junctions at Larger than tracer apical membrane epithelial (Peroxidase, junctions Mr=60,000 D) Obliterate and integrity extracellular space of epithelial junctions is Leaks greater around tight junctions Main function: Retards bulk flow of water Enters inter- across endothelium Resistance cellular spaces Endothelial junctions: 73±6 Ohms/cm2 Epithelial junctions: 1.6-9.1 k/cm2 (>1246X greater)  Nutritional transport occurs via paracellular route, i.e. solutes move between cells rather than being actively transported through them.  This type of transport requires that endothelium be leaky to substances within aqueous humor. Eye bank media to preserve human donor corneas Changes in corneal thickness of rabbit corneas during perfusion Rapid swelling Low pH Glutathione, bicarbonate Ringer’s (GBR) solution (Resembles aqueous humor) Maintenance of corneal thickness requires an intact barrier and metabolic pump Inhibits Na,K ATPase Inhibits carbonic anhydrase