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

1. 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. Oxygen
• Epithelium- dissolved oxygen from atmosphere into
tearfilm,through limbal capillaries
• Endothelium – Aqueous humour ( O2 tension- 72mmhg)
Glucose
• Corneal RQ – 1.0 , indicating glucose to be the prime source of
energy.
• Aqueous is the main source .
• Negligible amount comes from tear film and perilimbal capillaries
Amino
acids
• Aqueous is the main source – principally by passive diffusion
Sources of nutrients required for metabolism are:

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

23. 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.

24. 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.

25. 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

26. 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.

27. 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.

28. 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.

29. 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.

30. • 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.

31. 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.

32. 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.

33. 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.

34. Theories explaining
maintenance of Corneal
Avascularity
Chemical
Role of VIF
Role of VSF
Mechanical Combined

35. 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.

36. 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.

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

38. 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

39. 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.

40. 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 :

41. •Stromal Swelling Pressure (SP)
Factor which draw in
water
•Barrier Mechanism
Factor preventing inflow
of water
•Metabolic Pump
Factor which pump out
water from cornea
Evaporation • From the corneal surface

42. 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

43. • 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.

44. 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

45. 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.

46. 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

47. • 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.

48. • 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.

49. 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

50. 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

51. 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.

52. 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.

53. • 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.

54. 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

55. 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.

56. 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.

57. 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.

58. • 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

59. 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.

60. 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.

61. 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.

62. 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.

63. 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

64. 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

65. 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.

66. • 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

67. 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

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