PHYSIOLOGY OF CORNEA DETAILS

Physiology of cornea in which you will get all the details about corneal functions, corneal metabolism, wound healing and information about contact lenses

1. Physiology of cornea Ashish Neupane B. Optometry, NEH

2. Physiological Functions of Cornea To act as a powerful refracting media of fixed focus that transmits light in an orderly fashion for proper image formation. To protect the intraocular contents. In addition, the cornea also plays an important role in : - Absorption of topically applied drugs and - Wound repair after anterior segment surgery or trauma.

3. Biochemical and physiological processes concerned with the functioning of the cornea are as follows : Biochemical composition of cornea Metabolism of cornea Corneal transparency Drug permeability through the cornea Corneal wound healing

4. Biochemical composition of cornea Under normal conditions, biochemically cornea consists of approximately 78% water and 22% solids. 78% Water 22% solids

5. Solid components Collagen – 15% Type I - 50-55% Type III - <1% Type IV - 8-10% Type VI - 25-30% Other protein-5% Keratin sulphate -0.7% Chondritin /Dermatan sulphate -0.3% Hyaluronic acid and salts -1%

6. Epithelium Corneal epithelium constitutes 10% of total wet weight of cornea. Water - 70% of total wet weight Protein – Synthesis is 5x of stroma and 2x of Descemet’s membrane and endothelium. Lipids - 5.4% of dry epithelium - Mainly present in cell membrane - Phospholipids and cholesterol

7. Enzymes – Necessary for glycolysis, kreb’s cycle and active transportation. Electrolytes – Na ,K ,Cl Others - ATP – 2000 mmol /kg wet weight - Glutathione – 75-180 mg/gm - Ascorbic Acid – 47-94 gm/100gm - Ach - Cholinesterase

8. Stroma Main bulk of cornea – 90% of total thickness Water- (75%-80%) Solids-(20%-25%)

9. Anterior Stroma Posterior Stroma Obliquely oriented lamellae Less water content Less glucose More dermatan sulphate so less water absorption. Lamellae at right angle More water content More glucose More keratan sulphate so more water absorption

10. Solid components of corneal stroma Extracellular collagen Soluble Protein Proteoglycans Enzymes Matrix metalloproteinases Electrolytes

11. Soluble Proteins 5% of total wet weight of stroma Mainly consists of - Albumin - Immunoglobulin - Glycoproteins High level of IgA , IgG , IgM. Probably derived from serum and diffuse into the centre from limbus.

12. Extracellular Collagen Predominantly type I collagen. Type V , VI ,XII, XIV also present. Fibrills or lamellae – embedded in proteoglycans matrix. Diameter and spacing between fibrils are remarkably constant. Boiling water and acid – converts into Gelatin

13. Proteoglycans 4-4.5% of dry weight of cornea Three major fractions : - Keratan sulphate - Chondroitin sulphate - Chondroitin Present in interfibrillar space of stroma

14. GAGs are highly hydrophilic –important role in maintenance of corneal hydration level and transparency and ‘STROMAL SWELLING PRESSURE’. Mucopolysaccharidosis - abnormally high GAG in stroma.

15. Enzymes Stromal keratocytes – Glycolytic and kreb’s cycle enzymes Usually enzymatic activity is very slow when compared to the epithelium. Electrolytes Sodium content is high whereas potassium content is very low. Diffusible cations > Diffusible anions

16. Matrix Metalloproteinases MMP- family of enzymes that breakdown extracellular matrix components. Physiological role : - Maintenance of normal corneal framework - Remodelling after injury Source : - Resident cells during housekeeping function - Infiltrating inflammatory cells during pathological condition. - Secreted as pro-enzyme – Activated by cleavaging of a peptide from their N-terminal end.

17. Descemet’s Membrane Contents – Collagen (73%) and Glycoproteins Unique collagen structure: - Lacks typical 640-A band fibrils - Higher content of Glycine , Hydroxyglycine , Hydroxyproline . Doesn’t contain GAGs as cementing substance. Clinico -pathological implication : - Insoluble except strong acid or alkali - Extremely resistant to chemical and enzymatic (collagenase) action.

18. Endothelium Histochemical analysis shows – presence of essential metabolic enzymes i.e. Glycolysis and Krebs cycle.

19. Metabolism of cornea Metabolism is mainly required to produce energy for the maintenance of : -Transparency -Relative state of dehydration Most active part – Epithelium Second most active part - Endothelium

20. Sources of nutrients required for metabolism are:

21. Glucose Glucose is metabolized in the cornea by 3 metabolic pathway. Anaerobic glycolysis Glucose = Lactic acid + 2ATP Krebs cycle Glucose = CO2 + Water + 36ATP Hexose monophosphate shunt Glucose = NADPH + H2O + CO2 + 6ATP

22. Lactic acid Only 12% Glucose metabolised through krebs cycle. Rest converted to lactic acid. Not metabolized by cornea Removed by diffusion into aqueous humor Accumulation results in epithelial and stromal edema . Hypoxia doubles lactic acid concentration resulting in an osmotic gradient.

23. Corneal Transparency The main physiological function of the cornea is to act as a major refracting medium, so that a clear retinal image is formed. Maintenance of corneal transparency of high degree is a pre-requisite to perform this function. Normal corneal transparency is the result of anatomical and physiological factors.

24. Maintenance of Transparency Physical/Anatomical factor - Optically smooth tear film - Uniform and regular arrangement of non-keratinized epithelium - Peculiar arrangement of stromal lamellae - Uniform refractive indices of all layers - Avascularity - Absence of myelin sheath around corneal nerves Physiological factor - Relative state of dehydration

25. 1. Pre-Corneal Tear Film Forms an optically smooth and homogenous layer over anterior surface of cornea. Fills up small irregularities of corneal surface. Conditions associated with pre-corneal tear film results in loss of corneal transparency.

26. 2. Corneal Epithelium Normal epithelium is transparent due to the homogenicity of its refractive index. The basal cells of anterior epithelium are attached to the other neighbourhood cells i.e. laterally other basal cells and anteriorly wing cells by desmosomes and maculae occludentes .

27. 3. Arrangement of Stromal Lamellae Collagen fibrils of stroma bundled together in the form of lamellae. Arranged parallel to each other as well as to the surface. Two theories has been proposed : - Maurice theory - Theory of Goldman et al.

28. Maurice Theory David Maurice, Ph.D. – 1957 Cornea is transparent because the uniform collagen fibrils are arranged in a regular lattice so that the scattered light is destroyed by the mutual interference . As long as the fibrils are regularly arranged in a lattice, having less diameter (275-300 A) and separated by less than a wavelength of light (4000-7000 A), the cornea will remain transparent.

29. Loss of transparency will result, if this regular arrangement is altered by stromal oedema or mechanical stress. Electron Microscopy - Absence of lattice arrangement reported by some workers which is against the Maurice Theory.

30. Theory of Goldman et al Described originally by Goldman and Bendeck (1967) It nullifies the need of lattice arrangement to maintain transparency by diffraction theory . It postulated – fibrils are small in relationship to the light and will not interfere with light transmission unless they are larger than half of a wavelength of visible light i.e. 2000 A. Further confirmed by – ‘lakes’-areas devoid of collagen having dimension more than 2000A , in non-transparent human corneas. Similar ‘lakes’ can be found surrounding keratocytes in oedematous human corneas.

31. NOTE : The theory of Maurice as well as that of Goldman et al fail to explain the occurrence of rapid clouding of cornea associated with acute rise in IOP and rapid clearing of cornea with reduction of IOP.

32. 4.Avascularity of Cornea Cornea- avascular except for small loops which invade the periphery for about 1 mm. Pathological incidents leads to corneal vascularisation : -Invite defence mechanism against noxious agents. -Nutrition -Transport of drugs However, progressive corneal vascularisation is harmful –interference with functional properties of cornea.

33. Chemical Theory Role of VIF - Meyer and Chafre - Destruction of vaso inhibitory factors. Role of VSF - Campbell and Michaelson (1949) - Used experimental corneal burn - Release of VSF at the site of insult which diffuses through stroma upto the limbus and stimulates vascularisation. - Corneal hypoxia may also stimulate VSF release.

34. Mechanical Theory Cogan postulated – - Blood vessels cannot invade the normal cornea because of its structural compactness - Loosening of compactness of corneal stroma due to edema is mandatory for neovascularisation. Langhan postulation – - Neovascularisation occurs even in Fuch’s dystrophy and Aphakic bullous keratopathy. - Extension of edema upto limbus rarely produces vascularisation.

35. Combined Theory Demonstrated by Maurice et al. Release of VSF Structural loosening of compact corneal stroma by edema Neovascularisation

36. Superficial Vascularization Deep Vascularization Vessels originate from superficial limbal plexus Vessels are derived from anterior ciliary plexus Arranged in arborizing pattern Usually straight Present below epithelial layer Lie in stroma Continuity can be traced with conjunctival vessels Continuity cannot be traced beyond limbus Bright red in colour Pinkish in colour Types of corneal vascularization Deep Superficial

37. 5. Absence of myelin sheath around corneal nerves Corneal nerves loose their myelin sheaths at 1-2mm away from limbus. Thin and sheath-less nerves produces very little scattering of light.

38. 6. Relative state of corneal dehydration Cornea has the highest water content than any other connective tissue in the body i.e. 78% Four Factors are responsible for keeping the water content constant are :

39. From the corneal surface

40. Stromal Swelling Pressure The pressure exerted by GAG in the corneal stroma – 60 mm of hg (SP) These have an anionic effect on the tissue and therefore sucking the fluid with equal negative pressure = Imbibition Pressure (IP). Electrostatic repulsion of anionic charges of GAG mol expands tissue Sucking in of fluid with equal but negative pressure Imbibition Pressure

41. In-Vitro Imbibition pressure (IP) = Stromal Swelling Pressure (SP) In-Vivo IP changes with IOP IP =IOP-SP Therefore corneal edema is imminent when: IOP>SP In normal IOP, reduced SP SP has an interfibrillar tension causing the maintenance of normal fibril arrangement in the cornea.

42. Barrier Mechanism Barrier Mechanism is exerted by both epithelium and endothelium. Epithelium -Zonulae Occludentes -Desmosomes - Hemidesmosomes Endothelium -Not effective as epithelial barrier -Forms leaky channels allowing fluid to enter into stroma -Calcium dependent

43. Metabolic Pump It was initially suggested that water is actively transported into stroma by Fluid Pump. Later this postulation was nullified. Modern Theory -Water transportation occurs in association with ions – transported by different active pump mechanism Metabolic Pump -Proved by Temperature reversal Experiment.

44. Endothelial Metabolic Pump System Na/k ATPase pump system -Most active -Active extrusion of Na - Ouabian – specific ATPase inhibitor Bicarbonate dependent ATPase -Present in mitochondria, not on plasma membrane -Thiocyanate –specific inhibitor. -Essential for the maintenance of the corneal thickness

45. Carbonic Anhydrase Enzyme system - Produces bicarbonate and hydrogen ions - CA inhibitors – results in corneal edema further proving its role. Na/H Pump - This pump also has been postulated at the lateral plasma membrane surface. From the above , it is quite clear that a complex series of metabolically dependent reactions occur in the endothelium and epithelium to maintain proper fluid / ionic balance and deturgescence in the cornea.

46. Besides these systems, passive ion movement also occurs , in that K, Cl, and HCO3 ions diffuse into the aqueous humour. In the contralateral direction , Na, Cl, and HCO3 passively diffuse from the aqueous into the cornea.

47. Evaporation of water from the corneal surface Evaporation leads to increased osmolarity of precorneal tear film Hyperosmolarity of pre-corneal tear film draws in water from cornea Helps maintaining dehydration of cornea

48. 7 . Cellular Factors affecting transparency Keratocytes maintain transparency by producing collagens and proteoglycans. They contain enzymes involved in the assembly of stromal matrix. Specific enzymes defects are associated with corneal opacification E.g.Mucopolysaccharidoses

49. Drug Permeability Across The Cornea Topically instilled medications largely penetrate intraocularly through the cornea. Many factors which affect the drug penetration through the cornea are as follows : 1.Lipid and water solubility of the drug - Lipophilic property of corneal epithelium and endothelium that are crossed readily by non-polar (lipid soluble) drug - Hydrophilic property of stroma is easily crossed by polar (water soluble) compounds - Therefore, a drug should be amphipathic.

50. 2. Molecular size, weight and concentration of the drug Lipid soluble molecules can cross the corneal epithelium easily irrespective of their molecular size. Water soluble molecules with the molecular size less than 4A only can filter through the pores of the cell membrane. Molecular weight of less than 100 can pass readily through the cell membrane and those with more than 500 cannot Substances with large molecular size, when used in high concentration, then a small amount of drug can cross the cornea following laws of mass action.

51. The rate of penetration through the cornea of the drug depends upon their concentration in the solution. E.g. Pilocarpine, Homatropine , Atropine, Steroids. 3. Ionic form of the drugs Topical drugs must have capacity to exist in both ionized and non-ionized form for a better penetration through the cornea. Since non-ionized drugs can penetrate through the epithelium and the ionized drugs can pass through the stroma Due to the barrier of both the epithelium and stroma. Fluorescein which is negatively charged ion cannot penetrate the intact epithelium and this property forms the basis of fluorescein dye test.

52. 4. pH of the solution pH can also affect the penetration of the solutes by its effect on the electrical charges and stability of solutions. pH range between 4-10 doesn’t affect permeability pH of more than this range increases permeability 5. Tonicity of the solution Hypotonic solutions (those below 0.9% of sodium chloride) increase the permeability of the epithelium

53. Kinsey model of drug A typical model of the drug existing both in non-ionized and ionized form for penetration through cornea has been proposed by Kinsey for Homatropine .

54. 6. Surface active agents Agents that reduce the surface tension, increase corneal wetting and therefore present more drug for absorption. E.g. Benzalkonium chloride 7. Pro-drug form Pro-drugs are lipophilic and after absorption through the epithelium are converted into proper drug which can easily pass through stroma. For example, Dipivefrin is a pro-drug which is converted into epinephrine after its absorption into the eye. - Dipivefrin is more lipophilic than epinephrine and thus its corneal penetration is increased 17 times.

55. Cell Turnover And Wound Healing In The Cornea Epithelium Epithelium is constantly being regenerated by mitotic activity in the basal layer of cells. After epithelial debridement, the initial response of epithelium is to migrate as a flattened sheet of single cells across the stroma to close the defect. Hemidesmosomes and intracellular contacts then reform and gradually the single layer is restored to its six layered structure by mitosis in the peripheral basal cells.

56. Migration of epithelial cells is to achieved by marked cytoskeletal and cell shape changes involving redistribution of actin-myosin fibrils. Fodrin and E-Cadherin proteins precede the actin distribution in the cell. Migration of cells also dependent on intracellular signalling via components such as fibronectin, laminin and collagen peptides Adhesion of epithelium to the basement membrane and Bowman’s layer is achieved normally via hemidesmosomes , lamina densa and anchoring type VII collagen fibrils Most of the mitotic activity in the epithelium takes place at limbus

57. Stroma Incisional wounds of the cornea that involves the stroma may be accidental or intentional. Series of events in cornea to close the wound: - Deposition of fibrins within the stromal wound - Rapid epithelization of the wound incision - Activation of keratocytes to divide and synthesize collagen and GAGs. Loss of separation in the keratocytes such that they revert to a fibroblast like function Production of corneal matrix to restore clarity in small wound.

58. Endothelium Corneal endothelium doesn’t normally mitosis even after direct injury in a perforating corneal wound With age, there is a decrease in the number of cells with an increase in size and morphology. If sufficient amount of cells are lost then the cell layer cannot perform its pumping action and cornea decompensates water and becomes opaque.

59. Vascularization Vascularization occurs when vessels from the conjunctiva or the deep scleral plexus invade the periphery of the cornea during healing of the wound or corneal ulcers Cytokines, Macrophage Inflammatory Proteins(MIP) and Granulocytes-Macrophage Colony Stimulating Factor (GM-CSF) liberated from the inflammatory and local cells stimulate further ingress of inflammatory cells and initiate a vascularisation response.

60. Contact lens and cornea Predominantly affect the function of epithelium. Reduces the direct availability of oxygen to the epithelium. Increased lactate and carbon dioxide production.

61. Rigid Lenses Soft lenses Usually made from polymethylmethacrylate (PMMA) Depletes glucogen stores Induces inhibition of aerobic enzymes such as hexokinase reduces direct glucose utilization by cornea. Made from polymers of HEMA, poly-HEMA, vinylpyrrolidones , silicone, or other similar materials Permeable to oxygen and carbon dioxide Some degree of lactate accumulation

62. Complications in contact lens wearers Acute Chronic Epithelial thinning Hypoesthesia (decrease in normal sensation) Epithelial abrasions Stromal edema Endothelial blebs Corneal neovascularization Stromal thinning Corneal shape change alterations Endothelial cell polymegathism and pleomorphism

63. UV light filtration Cornea is more sensitive to UV light injury than the skin due to absence of melanin. Since human photoreceptors and corneal nerves cannot detect UV light, suprathreshold and repeated suprathreshold UV light injury can take place without the individual knowing it. This can cause acute photokeratitis of the epithelial surface or chronic irreversible keratopathies to the epithelium and anterior parts of corneal stroma. All UVC radiation is absorbed in the cornea due to high ascorbic acid content.

64. Cornea is the major filter of UV light for wavelength of 200-300 nm, protecting the lens and retina from damage. Amount absorbed Layer Absorbed by UVC 100% Epithelium Ascorbic acid UVB 90% Epithelium +Bowman’s layer Tryptophan, Ascorbic acid UVA 60% Partially attenuated Ascorbic acid

65. Cornea and ageing Epithelial basement membrane and Descemet’s membrane (from 3µm at birth to 13µm in adult) thickening Decreased keratocytes , sub-basal nerve fiber and endothelial cell density Increased stiffness and toughness of stroma Possible degeneration of extracellular matrix structures

66. References Anatomy and physiology of Eye, AK Khurana , 3 rd edition Jack J kanski , Brad Bowling, Clinical Ophthalmogy , 7 th edition