Scientific Basis of Vision Biochemistry- Iris and Lens Sep 02, 2009 Shiva Swamynathan

1. Scientific Basis of Vision Biochemistry- Iris and Lens Sep 02, 2009 Shiva Swamynathan Department of Ophthalmology University of Pittsburgh School of Medicine

2. List the components of the Uveal Tract and their relative location Iris (Anterior) Ciliary Body (Middle) Choroid (Posterior)

3. Where is the uveal tract attached to the sclera? Uveal tract is attached to the sclera at three points: 1. The sclera spur, 2. The exit points of vortex veins and 3. The optic nerve.

4. What is Iris? A highly pigmented tissue that functions as a diaphragm between the anterior and the posterior chambers of the eye, regulating the amount of light reaching the retina. What are the two chambers of the anterior compartment? The iris divides the anterior compartment, the space separating the cornea and the lens, into 2 chambers: 1. the larger anterior chamber (between the cornea and the iris), and 2. the smaller posterior chamber (between the iris and the lens).

5. What are the boundaries of the anterior and posterior chambers? Anterior Chamber- bounded by cornea, anterior iris and anterior lens surface. Posterior Chamber- bounded by ciliary body, lens and posterior iris. Note: this is not the vitreous cavity.

6. What is the basis for eye color? • Eye color comes from the iris (stroma in particular). When there is abundant melanin in the stroma and epithelial cells, the iris is brown; less melanin results in lighter shades of brown or yellow. Blue or green irides partly are the result of lack of melanin in the stroma combined with melanin in the epithelium and some other ill-defined structural change.

7. What are the different tissues in the iris? The Anterior endothelium consists of an incomplete layer of endothelial (mesothelial) cells on the anterior surface of the iris. The Irideal Stroma accounts for most of the mass of the iris and is composed of connective tissue, blood vessels and nerve fibers. Scattered within the stroma are numerous pigmented cells, usually containing melanin. The posterior boundary of the iris stroma, peripheral to the sphincter muscle, is demarcated by another sheet of smooth muscle, the dilator muscle. TheIrideal Epithelium is composed of two layers of densely pigmented cells.

8. What are Sphincter and dilator muscles? Sphincter muscle. The iris sphincter muscle encircles the pupil, and constricts the pupil in bright light or during accommodation. Dimensions: about 0.75 mm wide by 0.15 mm thick. Sphincter is controlled by parasympathetic nerve fibers. Dilator muscle. The dilator muscle fibers, arranged radially, extends from the base of the iris to the sphincter muscle. The fibers of the dilator muscle are derived from, and remain in continuity with, the cuboidal pigmented cell bodies in the iris pigment epithelium. Dilator muscle is controlled mainly by sympathetic nerve fibers, although some parasympathetic fibers are associated with it.

9. What is miosis? Constriction of the pupil (Narrow pupil) What is mydriasis? Relaxation of the pupil (Dilated pupil)

10. What is the effect of miotic agents and mydriatic agents on sphincter and dilator muscles? Miotic agents - Cholinergic agonists stimulate the sphincter and adrenergic blockers block the dilator Mydriatic agents – Cholinergic blockers block the sphincter and adrenergic agonists stimulate the dilator.

11. What is the source of blood supply to the iris? The iris is supplied from the major arterial circle in the ciliary body. The blood vessels of the iris run in a radial direction. The anterior border layer contains very few vessels. Iris blood vessels appear sheathed and have a characteristic loose appearance.

12. How is the iris innervated? The iris is supplied by the long and short ciliary nerves. The ciliary nerves pierce the sclera around the entrance of the optic nerve, and run forward in the perichoroidal space. After reaching the iris the ciliary nerves form a plexus around its attached margin. Nerve fibers ending in the anterior surface of the iris, Sphincter and Dilator are derived from these. The fibers derived through the motor root of the ciliary ganglion from the oculomotor nerve supply the Sphincter, while those derived from the sympathetic supply the Dilator.

13. What is the ciliary body? A structure connected to the iris and sclera, which produces aqueous fluid that fills the anterior compartment, thus maintaining the eye pressure. The ciliary muscle helps in focusing by facilitating the lens accommodation.

14. What is the main pharmacological target in treating glaucoma? Why? Ciliary body is the main target for treating glaucoma. It is rich in adrenergic, cholinergic and prostaglandins receptors, and their respective signal transduction pathways.

15. What germ-layer is the source of iris and ciliary body smooth muscle? Neuroectoderm (Unlike the smooth muscle elsewhere in the body, which is derived from the mesoderm)

16. What are the main functions of the iris and ciliary body smooth muscle? • Contraction/relaxation, • Receptor characterization, • Second messenger formation and regulation • Arachidonic acid release and eicosanoid biosynthesis

17. What are the broad classes of proteins expressed in the ciliary body? 20% - Protein synthesis, folding, secretion and degradation 12% - Energy supply and biosynthesis 6% - Cytoskeletal structure and contractility 7% - Cellular signaling and cell cycle regulation 2% - Nerve cell related 37% - Unknown

18. What is the rate of aqueous humor production in humans? About 2 ml/min. What are the components of the aqueous humor? Growth factors Enzymes such as carbonic anhydrase, lysozyme, diamine oxidase, dopamine hydroxylase, phospholipase A2, plasminogen activator, cAMP, prostaglandin, catecholamines, steroid hormones and hyaluronic acid.

19. How does aqueous humor enter the posterior chamber? Actively or passively? By both means. Actively, by means of energy dependent secretion including carbonic anhydrase-II activity. Passively, by diffusion and ultrafiltration. Active secretion accounts for a major amount of aqueous humor.

20. Describe the aqueous fluid outflow path The aqueous fluid produced by the ciliary body flows between the iris and lens, through the pupil to the drainage angle at the junction of the iris and the cornea. Aqueous fluid exits the eye through the trabecular meshwork and Schlemm’s canal, interscleral channels and episcleral vein. Aqueous humor is rich in macromolecules. True/False. False. Aqueous humor is practically free of proteins and other macromolecules, allowing for optical clarity. For example, it contains about 1/500 of the albumin present in the plasma.

21. What are Eicosanoids? Eicosanoids are signaling molecules with hormonal activity, made by oxygenation of twenty-carbon essential fatty acids. They exert complex control over inflammation or immunity, and serve as messengers in the central nervous system. They include compounds such as prostaglandins, prostacyclins, thromboxanes, and leukotrienes. Eicosanoids derive from either omega-3 (ω-3) or omega-6 (ω-6) EFAs. The ω-6 eicosanoids are generally pro-inflammatory; ω-3's are much less so. Anti-inflammatory drugs such as aspirin and other NSAIDs act by downregulating eicosanoid synthesis.

22. Which of the following statements about prostaglandin synthesis is true? • In response to cytokines, neurotransmitters or pharmacologic treatments, phospholipase-A2 is activated, releasing arachidonic acid from membrane phospholipids. • Free arachidonic acid is converted by cyclooxygenase-I (Cox1) or Cox2, to prostaglandin H2 endoperoxide intermediates. • Free arachidonic acid can also be metabolized through 5’-lipoxygenases and cytochrome P-450 pathways to generate leukotrienes and epoxides, respectively. • Phospholipase A2 can be inhibited by corticosteroids; Cox1 by non-steroidal anti-inflammatory drugs (NSAIDS), and the 5-lipoxygenase pathway by nordihydroguaiaretic acid (NDGA). • All of the above. Answer- E

23. True or False? • Prostaglandin analogs enhance outflow rather than formation, of aqueous humor. • b-blockers, CA-inhibitors and a2 agonists decrease the formation of aqueous humor. • NSAIDs bind irreversibly to cyclooxygenases blocking biosynthesis of prostaglandins from arachidonic acid. • COX1 is widely and constitutively expressed. • COX2 is expressed at low levels in normal physiologic conditions and upregulated in response to pro-inflammatory signals. • COX2 inhibitors (Vioxx, Celebrex, Bextra, etc) increase the risks of cardiovascular toxicity and complications. • Prostaglandin receptors are G-protein coupled 7-transmembrane domain containing membrane proteins • All of the above are true

24. What is the effect of Carbonic Anhydrase-II inhibitors on aqueous flow? CA-II inhibitors reduce the rate of entry of sodium and bicarbonate into the aqueous, thus reducing the aqueous humor formation.

25. What are the functions of the blood-aqueous and blood-retinal barriers? They control the composition and amounts of all material entering and leaving the eye. Only exception is the material that leaves the eye through Schlemm’s canal. When these barriers are defective, blood constituents mix with ocular fluids, causing plasmoid aqueous, retinal exudates or retinal edema.

26. What is the lens? What keeps it in place? • The lens is a transparent soft encapsulated biconvex structure composed of crystallins. • The lens is suspended by thin zonules that are attached to the ciliary body. • What is the size of the lens at birth and in adults? • The average anterior-posterior human lens thickness at birth is 3.5 - 4 mm and 4.5 - 5 mm after 65 years of age. • The diameter of the human lens at birth is 6.0 - 6.5 mm and 9.0 - 9.5 mm after 65 years of age. • The anterior surface of the lens has a greater radius of curvature than the posterior surface.

27. True/False • The lens is avascular and nourished by diffusion from the aqueous and vitreous. • The lens capsule is thicker at the anterior, compared to the posterior of the lens. • The radius of curvature of the anterior surface averages 10 mm, and it is subject to marked changes during accommodation. • Lens epithelial cells are metabolically active and regulate the water and ion balance of the entire lens. • Elimination of cellular organelles is necessary to reduce scatter of light passing through the lens. • The lens capsule allows free diffusion of water, ions, and small molecules, while acting as a barrier to large proteins such as serum albumin. • Adult lens is surrounded by a single celled epithelial layer. • Mature fibers support active transcription. • Central epithelial cells are mitotically active.

28. What is the lens capsule? What is it made of? The lens capsule is a 10-20 µm thick elastic basement membrane that completely envelopes the lens. It consists of hyaline material containing type IV collagen, small amounts of other collagens, glycosaminoglycans, laminin, fibronectin, and heparan sulfate proteoglycan. What is the lens epithelium? The lens epithelium is a single-celled layer of large cuboidal epithelial cells, beneath the anterior lens capsule. The cells of the lens epithelium continue to divide with age; thus, the lens continuously grows till death. The lens epithelial cells differentiate into fiber cells at the equator. The posterior capsule does not have any epithelial cells associated with it.

29. What is the structure of the lens cortex and nucleus? The adult lens can be divided into the cortex or the outer fibers laid down after the age of about 20 years, and the internal nucleus, consisting of cells produced from embryogenesis through adolescence. New lens cells are added to the margin of the lens throughout life from the lens epithelium. The cells at the lens nucleus do not undergo turnover or replacement and are among the oldest cells in the body of an adult.

30. What is the structure of the lens zonule? The lens zonule suspends the lens and holds it in place. The zonular fibers extend from both the anterior and posterior capsule of the lens to join the span fibers of the ciliary body and insert on the pars plana and retina.

31. What are lens sutures? As the differentiating fibers become fully elongated, they meet with fibers that have elongated from the other end, making the lens sutures. Name the major lens-specific integral membrane protein. Aquaporin-0 or Major Intrinsic Protein (Aqp0 or MIP). What are the abundant water-soluble proteins in the lens called? Crystallins. Alert- There is a wrong statement about the crystallins in section 2 of BCSC. In page 275, it states that crystallins constitute 90-95% of total lens protein. It is just not true. It should be as high as 90-95% of water-soluble proteins, and not total proteins.

32. What do lens fiber cell membranes contain? • Tight packing and low fluidity in the mature lens fiber cell membranes is achieved by • High amount of saturated fatty acids, • High cholesterol:phospholipid ratio, and a • High concentration of sphingomyelin • Lipids contribute about 1% of total lens mass

33. Name two requirements for proteins to be recruited as crystallins. • Should be water-soluble, without precipitating at high concentrations • Should be stable and stay active for a long time • What are the two general groups of crystallins? • Ubiquitous Crystallins (Present in all vertebrates) • e.g.,a-Crystallin and b/g-crystallins • Taxon-specific crystallins (Different in different species) • e.g., e-crystallin • What is the native molecular mass of a-crystallin? • Larger than 600kDa; can be as high as 1500kDa. • aA- and aB-crystallins are both around 20kDa monomers. • a-crystallin is a super-aggregate of these monomers.

34. How is spherical aberration avoided by the lens? Glass bead in water Fish lens in water Decreasing gradient of refractive index from the centre to the periphery of the lens resulting from continual lens development minimizes the spherical aberration

35. True/False • b- and g-crystallins are structurally related. • True • aB-crystallin is a widely and constitutively expressed member of the small heat shock proteins family and is inducible by heat and other forms of stress. • True • g-crystallin tends to be concentrated in the nuclear region of the lens, as it is abundantly expressed early in development. • True • Most taxon-specific crystallins are oxidoreductases which bind pyridine nucleotides. Reduced nucleotides absorb UV light, protecting the retina from oxidative damage. • True • In the normal lens, concentrations of sodium are low (~10mMol/L) and potassium, high (~120mMol/L) relative to aqueous humor, which contains high sodium (~150mMol/L) and low potassium (~5mMol/L). • True • There is a smooth gradient of refractive index in the lens, with the oldest cells in the center having the lowest refractive index and the newest cells in the periphery having the highest refractive index. • False • aA-crystallin has chaperone-like activity, which is absent in the aB-crystallin. • False

36. What happens when lens proteins aggregate? • Results in cataract- scatter of light, loss of transparency. Thus, chaperone activity of a-crystallin is crucial for long-term maintenance of lens transparence. • List two metabolic causes of cataract. • Sugar cataract caused due to diabetes mellitus • Sugar cataract caused due to defective galactose metabolism.

37. What maintains the ionic balance in the lens? • A sodium-potassium ATPase pump, an intrinsic membrane protein that hydrolyzes ATP, to transport Na+ out and K+ in to the lens. • Na+-K+-pumps are found primarily in the anterior surface of the lens, in the epithelium and outer, immature fiber cells. • How do lens cells communicate with each other? • Through gap junctions consisting of connexin 43 in the epithelial cells and Cx-46 and -50 in the fiber cells. • MIP (Aqp0) also helps in intercellular communication. • What is the primary source of energy in the lens? • Anaerobic glycoslysis is the primary source of energy in the anterior lens. Pentose phosphate pathway is used in oxidative stress conditions, to replenish NADPH.

38. Questions/Comments? Room 1025, EEI Phone: 412-802-6437 Swamynathansk@upmc.edu

40. Genes involved in iris development