Understanding the Anatomy and Function of Iris, Ciliary Body, and Lens in Vision

1. Scientific Basis of Vision Iris, Ciliary Body and Lens Shiva Swamynathan Department of Ophthalmology University of Pittsburgh School of Medicine

2. Uveal Tract – Vascular middle layer of the eye Iris (Anterior), Ciliary Body (Middle), Choroid (Posterior) Iris: Pigmented diaphragm located between anterior and posterior chambers. Regulates the amount of light reaching the retina. Ciliary Body: Regulates the composition and production of aqueous humor, affecting the ionic environment and metabolism of the lens, iris, TM and cornea. Choroid: Vascular layer of the eye located between the retina and the sclera. Provides nourishment and oxygen to outer layers of the retina.

3. Iris - Anatomy AC Sphincter Anterior Border Stroma Dilator Pigmented epithelium Lens PC Anterior Endothelial Border- An incomplete layer of endothelial (mesothelial) cells. The Irideal Stroma accounts for most of the mass of the iris. Composed of connective tissue, blood vessels, nerve fibers and scattered pigmented cells containing melanin. Dilator and Sphincter muscles- Regulate iris dilation.Sphincter encircles the pupil. Dilator, a smooth muscle, is in the posterior stroma peripheral to the sphincter. Thepigmented epithelium is composed of two layers of densely pigmented cells, and is continuous with the ciliary epithelium.

4. Iridial Muscles- Sphincter and Dilator (derived from neural ectoderm) Sphincter muscle. Encircles the pupil, and constricts the pupil in bright light or during accommodation. Controlled by parasympathetic nerve fibers. Dilator muscle. Arranged radially, these fibers extend from the base of the iris to the sphincter. Controlled by sympathetic nerve fibers, although some parasympathetic fibers are associated with it. Main functions of iris and ciliary body smooth muscle Contraction/relaxation Second messenger formation and regulation Arachidonic acid release and eicosanoid biosynthesis

5. Blood Supply to the Iris Source: 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.

6. Innervation of the Iris The iris is supplied by the ciliary nerves, which pierce the sclera around the optic nerve and run forward in the perichoroidal space. After reaching the iris the ciliary nerves form a plexus around its attached margin, from which the nerve fibers ending in the anterior surface of the iris, Sphincter and Dilator are derived.

7. Ciliary Body • Produces aqueous fluid that fills the anterior chamber, maintaining IOP • The ciliary muscle facilitates lens accommodation Pigmented epithelium is leaky while non-pigmented epithelium facing the aqueous is responsible for the blood-aqueous barrier In humans, aqueous humor is produced at a rate of about 2-3 ml/min

8. Macromolecules in Aqueous humor Aqueous humor contains few macromolecules, ensuring optical clarity. e.g., albumin concentration is about 1/500 of that in the plasma. Entry of aqueous humor into the posterior chamber Actively - Energy dependent secretion carbonic anhydrase-II activity dependent. Passively- Diffusion and Ultrafiltration. Active secretion accounts for the bulk of aqueous humor

9. Aqueous fluid outflow Trabecular pathway - major path for aqueous outflow. Aqueous flows between the iris and lens, through the pupil to the drainage angle at the junction of the iris and the cornea, exiting the eye through the trabecular meshwork and Schlemm’s canal, interscleral channels and episcleral vein. Uveoscleral pathway - accounts for ~ 20-40% of the outflow. Flows through the ciliary body and iris root to the ciliary muscle and suprachoroidal space to veins in the ciliary body, choroid, and sclera.

10. Eicosanoids Signaling molecules with hormonal activity, made by oxygenation of 20-carbon essential fatty acids. They control inflammation and serve as messengers in the CNS. They include compounds such as prostaglandins, prostacyclins, thromboxanes, and leukotrienes. Eicosanoids are derived from either omega-3 (ω-3) or ω-6 EFAs. Unlike the ω-3's, ω-6 eicosanoids are generally pro-inflammatory. Anti-inflammatory drugs such as aspirin and other NSAIDs act by downregulating eicosanoid synthesis. Eicosanoids play critical roles in regulating aqueous outflow.

11. Prostaglandin Synthesis • In response to cytokines, neurotransmitters or pharmacologic treatments, phospholipase-A2 is activated, releasing arachidonic acid (AA) from membrane phospholipids • Free AA is converted to prostaglandin H2 endoperoxide intermediates by cyclooxygenase-I (Cox1; ubiquitously expressed) or Cox2 (expressed in pro-inflammatory conditions) • Free AA can be metabolized through 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); lipoxygenase pathway by nordihydroguaiaretic acid (NDGA).

12. Modulating aqueous production and outflow • Prostaglandin analogs enhance outflow rather than blocking the formation of aqueous humor. • CA-inhibitors, b-blockers and a2 agonists decrease the formation of aqueous humor. • NSAIDs bind irreversibly to Cox1 blocking prostaglandin biosynthesis • 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. 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.

13. Neurotransmitters and Receptors in the Iris and Ciliary Body Muscle Receptor Agonists Sphincter Cholinergic Ach, Muscarine (&Ciliary) Nicotine Dilator Adrenergic Norepinephrine, Phenylephrine… • Sphincter and ciliary muscles contain cholinergic muscarinic type receptors and are innervated by the parasympathetic third cranial oculomotor nerve • Dilator muscles contain a-adrenergic type receptors and are innervated by sympathetic nerves from the superior cervical ganglion. Adrenergic impulses are transmitted to the muscles by norepinephrine • Sensory neurotransmitters like substance P and calcitonin gene-related peptide (cgrp) may regulate inflammatory reactions and irideal muscle tone

14. Effect of miotic and mydriatic agents on iris and ciliary muscles Muscle Receptor Agonists Sphincter Cholinergic Ach, Muscarine (&Ciliary) Nicotine Dilator Adrenergic Norepinephrine, Phenylephrine… Miotic agents Cholinergic agonists stimulate the sphincter (Constrict the pupil) and result in increased accommodation. Adrenergic blockers block the dilator. Mydriatic agents Adrenergic agonists stimulate the dilator (Dilate the pupil) Cholinergic blockers block the sphincter and act as cycloplegics by blocking ciliary muscles.

15. The choroid • Posterior segment of the uveal tract, located between retina and sclera • Provides nutrients and oxygen to the outer layers of the retina • Blood from the choroidal vessels drains via the four vortex veins • Choroid is bounded by Bruch's membrane and the sclera Retinal Pigment Epithelium Bruch’s Membrane Choriocapillaris Medium Choroidal Vessels Large Choroidal Vessels Suprachoroidal space Sclera

16. The Lens Crystalline, transparent, circular and biconvex structure located behind the iris between the aqueous and the vitreous, that helps in accommodation and refraction Two Important functions of the lens: Accommodation and Refraction. Accommodation –When we focus on a nearby object, the ciliary muscle in the suspensory ligaments contracts, releasing the tension on the lens capsule and the anterior surface of the lens becomes more curved or more convex. This increase in curvature of the anterior surface of the lens helps focus the divergent rays coming from a near object onto the retina. Refraction - The lens has a refractive power of about 15-16 dioptres, contributing to about a fourth of the total refractive power of the eye

17. The Lens Lens capsule: thin, transparent collagenous sac covering the lens. Thicker in the anterior. Anterior epithelium: a single layer of cuboidal cells that progressively differentiate as they migrate towards the equator to become lens fiber cells. Lens fiber cells: Protein-filled cells lacking nucleus and organelles compactly arranged to form the core of the lens. Newer fibers are progressively deposited over older ones so that the nucleus at the very core of the lens contains the oldest fibers. Lens nucleus consists of the central fiber cells produced during embryonic to adolescence stage Lens cortex consists of the outer fibers laid down after adolescence. New fiber cells are added to the lens margin throughout life

18. The Lens • 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 light scatter. • The lens is avascular and nourished by diffusion • A normal lens contains low sodium (~10mM) , and high potassium (~120mM) relative to aqueous humor, which contains high sodium (~150mM) and low potassium (~5mM).

19. Lens Development

20. What keeps the lens in place? • The lens is suspended by thin zonules that are attached to the ciliary body. • The lens keeps growing throughout life • 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.

21. Lens fiber cell membranes • Lipids contribute about 1% of total lens mass • Tightly packed membranes with low fluidity • High amount of saturated fatty acids • High cholesterol:phospholipid ratio • High concentration of sphingomyelin • The most abundant lens-specific integral membrane protein is aquaporin-0 or Major Intrinsic Protein. Aqp0 (MIP) helps in solute transport through the lens. MIP

22. Ionic Balance in the Lens • A sodium-potassium ATPase pump, an intrinsic membrane protein hydrolyzes ATP, to transport Na+ out and K+ into the lens. • Na+-K+-pumps are found primarily in the anterior surface of the lens, in the epithelium and outer, immature fiber cells. • Intercellular Communication in the Lens • 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. • Primary Source of Energy in the Lens • Anaerobic glycolysis is the primary source of energy in the lens. • Pentose phosphate pathway is used in oxidative stress conditions, to replenish NADPH.

23. Lens Crystallins Abundant, stable and water-soluble proteins responsible for the transparent and refractive properties of the lens • Two critical properties of lens crystallins • Should remain water-soluble at high concentrations • Should be stable and stay active for a long time • Two classes of crystallins • Ubiquitous (Present in all vertebrates) • e.g.,a-Crystallinandb/g-crystallins • Taxon-specific (Different in different species) • e.g., e-crystallin • Crystallins exist as huge macromolecular aggregates • aA- andaB-crystallinsare both around 20kDa monomers. • a-crystallin is a super-aggregate of these monomers, often larger than 600kDa; can be as high as 1500-2000kDa.

24. Lens Crystallins • aA- and aB-crystallins are members of the small heat shock protein family, and exhibit chaperone-like activity • aA-crystallin is expressed in a lens-preferred manner. In contrast, aB-crystallin is a widely and constitutively expressed and is inducible by heat and other forms of stress • b- and g-crystallins are structurally related. g-crystallin tends to be concentrated in the nuclear region of the lens, as it is abundantly expressed early in development • Most taxon-specific crystallins are oxidoreductases which bind pyridine nucleotides. Reduced nucleotides absorb UV light, protecting the retina from oxidative damage • The lens displays a smooth gradient of refractive index, which is highest in the oldest cells in the center and the lowest in the newest cells at the periphery

25. Lens is designed to avoid spherical aberration 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

26. Pop Quiz

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

28. 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. • What is the basis for eye color? • Eye color comes from the iris. Abundant melanin in iridial stroma and epithelial cells leads to a brown iris; less melanin results in lighter shades of brown or yellow. Blue or green irises are the result of lack of melanin in the stroma combined with melanin in the epithelium and some other ill-defined structural change.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. What are the two main functions of the ciliary body? Ciliary body, connected to the iris and sclera, produces aqueous fluid that fills the anterior compartment, maintaining the eye pressure. The ciliary muscle facilitates lens accommodation

35. 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 associated signal transduction pathways. 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.

36. What is the rate of aqueous humor production in humans? About 2-3 ml/min. 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.

37. Describe the major outflow path for aqueous fluid 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. True/False: Aqueous humor is rich in macromolecules. 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.

38. 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.

39. 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

40. 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

41. 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.

42. 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 lens fibers support active transcription. • Central epithelial cells are mitotically active.

43. 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 Crystallins? Abundant water-soluble proteins in the lens (also in cornea) 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

44. What keeps the lens in place? • The lens is suspended by thin zonules that are attached to the ciliary body. • What are the features of the lens fiber cell membranes? • Tightly packed with fairly low fluidity • High amount of saturated fatty acids • High cholesterol:phospholipid ratio, and a • High concentration of sphingomyelin • Lipids contribute about 1% of total lens mass

45. 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.

46. 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

47. 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 • Normal lens contains low sodium (~10mM) and high potassium (~120mM) relative to aqueous humor, which has high sodium (~150mM) and low potassium (~5mM) • 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

48. 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.

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