Sanket Parajuli

CORNEA

Embryology

• Dev. of the cornea is triggered by the separation of the lens vesicle from the surface ectoderm

• Epithelium : surface ectoderm 40days

• Stroma (7 weeks) and bowman’s membrane (4 month): mesenchyme

• Endothelium (40days) : neural crest cells

• Descemet’s membrane(4th month) : endothelium

CORNEA

• Transparent avascular tissue with a smooth, convex surface and concave inner surface

• Forms the principal refractive surface, accounting for 70% (40-45 diopters) of the total refractive power

• Most of the refraction of the eye occurs not in the lens but at the front surface of the cornea at the tear/air interface

• Refractive index of cornea = 1.37

• The curvature of the cornea is greater than that of the sclera so that a slight external furrow (scleral sulcus) separates it from the sclera. This furrow may be demonstrated in the living eye by specular reflection from the overlying tear film.

DIMENSIONS

• Anteriorly::: elliptical ..11.7 mm wide &10.6 mm high

• Posterior surface :: circular..11.7 mm in diameter

• This difference is due to the greater overlap of sclera and conjunctiva above and below than laterally

• The cornea forms part of what is almost a sphere, but it is usually more curved in the vertical than the horizontal meridian, giving rise to astigmatism 'with the rule'.

The axial thickness of the cornea is 0.52 mm with a peripheral thickness of 0.67 mm

Central 1/3rd of cornea = optical zone

Radius of curvature of the anterior surface is about 7.8 mm and that of the posterior 6.5 mm, in adult males.

Peripheral cornea is more flattened

TOPOGRAPHY

• Corneal shape is important to the fitting of contact lenses

• Average anterior radius of curvature 7.2- 8.4

• The cornea is flatter in men than in women

• The anterior curvature of the cornea is spherical over a small zone 2-4 mm in diameter which is decentered upwards and outwards relative to the visual axis, but correctly centered for the pupillary aperture (which lies 0.4 mm temporally). This is sometimes termed the corneal apex or cap (Fig)

• The corneal curvature varies from apex to limbus. There is greater flattening nasally than temporally, and above than below, although variations occur

• These features influence the fitting of contact lenses

• The cornea flattens slightly on convergence

STRUCTUREBehind the precorneal film are five tissue layers :1. Epithelium2. Bowman's layer3. Stroma4. Descemet's membrane5. Endothelium

• Researchers revealed a new layer of tissue in the human cornea. • Named Dua’s layer, after Professor

Harminder Dua from the University of Nottingham in the U.K• Between the corneal stroma and the

Descemet’s membrane

Epithelium• stratified, squamous and non-keratinized • It is continuous with that of the conjunctiva at the

corneal limbus, but differs strikingly in possessing no goblet cells. • The epithelium consists of 5-6 layers of nucleated

cells a) Basal cells: stand in a palisade like manner in

perfect alignment on a basal lamina & form the germinative layer of the epithelium, continuous peripherally with that of the limbus. These basal cells are columnar with nucleus oriented parallel to the cell's long axis.

b) The second epithelial layer (the 'wing' or 'umbrella' cells) consists of polyhedral cells, convex anteriorly, which cap the basal cells, and send processes between them. The long axes of their oval nuclei are parallel to corneal surface

c) The next two or three layers are also polyhedral and become wider and increasingly flattened towards the surface. (surface cells)The surface cells have the largest surface area and this is greater in the periphery compared to centrally

Ultrastructural features

• Mitochondria: scarce --basal cells but abundant --wing and middle cell layers

• High glycogen content in the form of granules, especially in the wing and superficial cells

• Amount of glycogen falls in hypoxic conditions, or during wound healing

• The basal cells are connected to one another by desmosomes and to basal lamina by hemidesmosomes

• In basal cells , anchoring filaments pass through the hemidesmosomal structures to be inserted into the underlying basal lamina.

• This is a strong attachment and if the corneal surface is scraped by a scalpel blade, fragments of the ruptured basal cells remain attached to the basal lamina

• Between contiguous epithelial cells, in addition to the desmosomal connections, tight junctions (zonulae occludentes) run circumferentially between contiguous surface cells

• These tight junctions are relatively impermeable to small molecules such as sodium ions and makes the epithelium semipermeable

• The most superficial cells of the epithelium exhibit surface microvilli or microplicae

• Microvilli serve a physical function in stabilizing the deep precorneal tear film

• Scanning microscopy also demonstrates 'light' and 'dark' cells with varying density and type of microvilli present

• It has been suggested that the dark cells are older and about to

desquamate

• Langerhans cells have been identified in the corneal epithelium( in the peripheral region)

• These cells and OR positive macrophages are almost totally absent from the central cornea but will populate this region in response to infection.

Basal lamina

• Basal lamina is secreted by the basal cells, which also synthesize the hemidesmosomal structures concerned in attachment of epithelium to the lamina

• The lamina consists of collagen and glycoprotein

constituents integrated structurally with the underlying Bowman's layer, to which it is firmly attached by an array of short anchoring filaments

• Derma epidermal junction of cornea/conjunctiva is similar as of skin

• Structures present in basal lamina:a) lamina lucida b) lamina densa c) anchoring fibrils & anchoring plaques :electron-dense fibrils form narrow bundles which insert into the subjacent stroma or Bowman's laver

• These structures are composed of type VII collagen(lamina densa contains type IV collagen)

• The cohesion between the basal lamina and Bowman's zone …loosened by lipid solvents, stromal oedema and inflammation but it remains attached to the basal cells

• The basal lamina may be destroyed by proteolytic enzymes such as trypsin and chymotrypsin

• With old age, and in diabetes, it becomes thickened and multilamellar

Metabolism

• The corneal epithelium is rich in glycogen, which serves as an energy store in the aerobic conditions .. (With the eyes open, the tear PaO2 is 155 mmHg; With the eyes closed this drops to about 55 mmHg)

• In hypoxic conditions, such as those induced by a tight contact lens, the epithelial glycogen level falls.

• Hypoxia also produces a profound fall in corneal sensitivity

• The concentrations of acetylcholine and acetylcholinesterase in the corneal epithelium are as high as in brain tissue

• A role for acetylcholine in transport processes has been postulated or in regulating epithelial cell mitosis, because acetylcholine may stimulate cyclic GMP (cGMP) production, and cGMP stimulates epithelial cell mitosis

Turnover

• studies using titrated thymidine suggested that the epithelium was replaced approximately weekly by the division of basal epithelial cells

• One daughter cell from a division remained in the basal layer while the other was displaced to the surface, from where it was ultimately shed

• Germinative region of the corneal epithelium(stem cells), lies at the limbus.

• Cells migrate from the limbus towards the centre of the cornea

• limbal basal cells can be distinguished from corneal epithelial cells and from other limbal epithelial cells by their expression of cytokeratins

• corneal epithelial cells and suprabasal limbal epithelial cells express cytokeratins typical of differentiated cells (e.g. CK3)

• Basal limbal cells are negative for these cytokeratins and positive for a group of acidic cytokeratins staining with the antibody AEl

• on the basis of experimental evidence, that there was both a limbal basal and a corneal basal epithelial source for corneal epithelial cells (XYZ hypothesis)

• The sequence of events from proliferation of stem cells to desquamation of superficial corneal epithelial cells is thought to involve cell division by the slow cycling stem cells

Attachment• The basal epithelial cells are firmly attached to basal lamina by

hemidesmosomes and anchoring filaments

Bowman's layer (anterior limiting lamina)

• Narrow, acellular homogeneous zone, immediately subjacent to the basal lamina of the cornea epithelium

• Some observers believe that the epithelium has a role in the laying down and maintenance of Bowman's layer

• In pathological conditions (such as corneal edema or certain corneal dystrophies) and after death, the epithelium separates readily from this limiting layer.

Ultrastructural features• Bowman's layer consists of a meshwork of fine collagen

fibrils of uniform size, lying in a ground substance

• The compacted arrangement of the collagen confers great strength to this zone

• Bowman's layer is relatively resistant to trauma, both mechanical and infective; once destroyed it is not renewed but is replaced by coarse scar tissue

• It is perforated in many places by unmyelinated nerves in transit to the corneal epithelium .

Stroma (substantia propria)• Consists of collagen fibrils(lamellae) & cells in

groundsubstance (proteoglycans) Lamellae:Arranged in many layersNot only parallel to each other but also to corneal planeBecomes continuous with scleral lamellae at limbusOblique orientation in the anterior 1/3rd

Run @ right angles to one another in posterior 2/3rds

• Parallel arrangement of collagen fibrils of central cornea extend to periphery where fibrils adopt a concentric configuration to form a Weave(net like) @ limbus

• This gives peripheral cornea strength and helps maintain its curvature

• Parallel arrangement of fibrils make intralamellar dissection during superficial keratectomy and lamellar keratoplasty easy

Cells

Keratocytes , macrophages and lymphocytes are present in the stroma

Corneal keratocyte: are fibroblasts which are found throughout stroma mainly between the lamellae

Have flattened cell body with long processes

They produce ground substance

Stromal repair• Repair of the stroma involves keratocyte activation, migration and transformation

into fibroblasts and the production of scar tissue.• Larger wounds provoke a rapid vascular response in addition, with an invasion by

PMN leucocytes and monocytes. • Transformation of the monocytes into fibroblasts requires the presence of an

overlying epithelium.• Collagen fibrils are initially laid down without regularity, and are larger than in

normal cornea. • Remodeling of the scar tissue ensues, with thinning of fibrils, reformation of

lamellae over many months and an increase in transparency.• Lymphatic channels, normally absent from the cornea, appear in vascularized

scars and persist after injury.

Descemet's membrane (posterior limiting layer)

• Descemet's membrane is the basal lamina of the corneal endothelium

• strong resistant sheet @ the back of the corneal stroma

• unlike Bowman's layer, it is sharply defined and the plane of separation is used at lamellar keratoplasty.

• Descemet's membrane thickens with age and in degenerative conditions of the corneal epithelium such as congenital endothelial dystrophy or posterior polymorphous dystrophy.

• The major protein of Descemet's layer is type IV collagen.

Ultrastructural features• The laminated structure of Descemet's membrane has been demonstrated by several

workers

• membrane consist of superimposed flat plates forming a lamellar pattern of equilateral triangles . The triangles are interconnected by electron-dense nodes and internodes

• In the ageing cornea, bands of long-spacing collagen may be found in Descemet's membrane likely due to polymerization of the collagen

• Also, in the ageing cornea, focal overproduction of basal lamina like material produces peripheral growths called Hassal-Henle warts . These are fissured and show receding cytoplasmic invaginations on their endothelial faces

• Although they are said to be a part of 'physiological ageing process' by some, they resemble Descemet's warts of the central cornea (corneal guttata)

• The peripheral rim of Descemet's membrane is the internal landmark of the corneal limbus and marks the anterior limit of the drainage angle (Schwalbe's line)

• After traumatic interruption of Descemet's membrane and the endothelial layer (penetrating injury, birth trauma, or rupture in the acute hydrops of keratoconus), the endothelial layer will resurface the defect by spread of its cells and synthesis of fresh basal lamina structurally identical to normal Descemet's layer.

• This contrasts again with Bowman's layer, which is replaced by disorganized coarse fibrillar scar tissue after injury.

Endothelium

• The endothelium is a single layer of hexagonal, cuboidal cells --posterior to Descemet's membrane

• These cells differentiate from cells that migrate from the limbal area

• They are not vascular in origin

• After injury of any kind, damaged cells are replaced by a spreading of cells from adjacent zones, the cells increasing in area (up to three times their span) but decreasing in height

-Endothelial cells density –-about 6000 cells/mm² at birth-26% lost in 1st year-Further 26% lost over next 11 years-Rate of cell loss slows and stabilizes around middle age and then it is about 2500

cells/mm²-If cells density falls upto 500 cells/mm² corneal oedema develops and transparency

reduced• Adult: 2500-3000/mm2• 0.6% / year decrease of endothelial cell density• Corneal function is maintained @ 300-600/mm2• If endothelial cell count < 2000/mm2--------not used for transplant

-At birth cells are 10 μm in height, with age it becomes flattened to 3-5 μm and 18-20 μm width-Single oval nucleus located centrally-Cells shape is hexagonal in youth with age it become polymorphic polymegathism(change in size), pleomorphism(change in shape)

disruption of mosaic pattern

Blood supply of cornea:

• Avascular

• Devoid of lymphatic drainage

• Cornea nourished by AH and capillaries around cornea

• Center part of cornea receives O2 from air, dissolved in tear whereas peripheral part receives O2 from anterior ciliary vessels

NERVES OF THE CORNEA

• The cornea is supplied by the ophthalmic division of the trigeminal nerve via the anterior ciliary nerves and those of the surrounding conjunctiva

• Anterior ciliary nerves enter the sclera from the perichoroidal space a short distance behind the limbus. They connect with each other and with the conjunctival nerves, forming pericorneal plexuses at various levels.

• The nerves pass into the cornea as 60-80 flattened mainly myelinated branches surrounded by perineurium

• After about 1-2 mm in the stroma they usually lose their myelin sheaths and divide into two groups forming stromal plexus

anterior andposterior..

• The anterior nerves (40-50) pass through the substance of the corneal stroma and form a subepithelial plexus subjacent to the anterior limiting membrane

• The fibers then pierce pores in bowman’s membrane lose their schwann sheath divides into filaments under the basal layer of epithelium and extend between cells as intraepithelial plexus

• Thus cornea has extensive innervaton highest @center and decreasing @ periphery• No nerves are present in central posterior part of

cornea, DM and endothelium

Surgical anatomic zones

• For practical purposes the surface of the cornea can be divided into two general regions:

a) the central optical zone (3-4mm in diameter)b) the remainder of the cornea(paracentral, peripheral, limbal)

The central optical zone is the refractive region for forming the image on the foveal part of the retina.

The remainder of the cornea serves only as mechanical support and also optical importance in peripheral vision and when pupil is widely dilated.

Surgically 4 concentric anatomic zones are recognized.

Aging changes in cornea:

• Old age…cornea has translucent dust like opacities

• Bowman’s & DM…..increase in thickness

• Arcus senilis is present …infiltration of extracellular lipid

• Hassal Henle bodies: small protrusion at periphery of DM

Corneal physiologyBiochemical composition of cornea• 80% water & 20% solids ( 2% salts… rest organic matter)1) Epithelium: 10% of total wet weight of corneaWater Protein ( 5* higher than in stroma 2* higher than DM and endothelium)Lipids (phospholipids, cholesterol)Enzymes for glycolysis and krebs cycleAch and cholinesterasesElectrolytes( high concn of K+ nd low concn of Na)

• Epithelium has 2 superficial layer of flattened cells connected by tight junctions( Zona occludens)

• Acts as a barrier to prevent fluid movement from tears to stroma

• With removal of epithelium the stroma swells 150% than normal within 4-6 hours

• Epithelium maintained by mitosis in basal cells• Mitotic rate = 10-15% / day• Turnover time of epithelium : 7 days

XYZ hypothesis:• Corneal epithelium is maintained by migration of basal cells from limbal

epithelium

• Migration takes place from the basal limbal cells centripetally (initially form transient ampliftying cells and then form fully differentiated cells)

• Epithelium maintained by:a) Shedding of superficial cellsb) Mitosisc) Cell migration

2) Stroma: main bulk of cornea (90% of total thickness)• 75-80% water, collagen, other proteins, keratan sulfate and dermatan sulfateCollagen: type 1 most predominant(type 5,6,12,14 also present)• Corneal collagen (as other collagen) have high glycine, proline and

hydroxyproline content • In Acid burns corneal collagen converted into gelatin::acid burns less serious than

alkali burns Proteins: albumin, IgA, IgG and IgMProteoglycans: are glycosylated proteins with @least 1 GAG chain with a protein core• 3 major GAGs : keratin SO4, Chondroitin SO4, and chondroitin• GAGS are present in interfibrillar space thus accounts for “stromal swelling

pressure”—60 mm Hg (tendency to retain water—maintain corneal hydration)

Enzymes: Glycolytic and krebs enzymes present keratocytes…enzyme activity in stroma is low than in epitheliumMatrix metalloproteinases:Family of enzymes that breakdown ECMHelp maintain framework of cornea and have crucial role in remodeling after injury

Electrolytes : High Na+ content low K+ compared to epithelium

• Anterior stroma : less water than posterior stroma (anterior=3.14gm H2o/gm dry weight posterior 3.85gm H2o/gm dry weight)

• Anterior stroma less glucose than posterior

• Dermatan sulfate more anteriorly (DS has more water retension property)• Keratan sulfate more posteriorly (KS has less water retension property)• most edema occurs in posteriorly

• Greater corneal swelling when endothelial barrier removed compared to epithelial barrier

3)Descemet’s membrane: collagen(73%) and glycoproteinsNo GAGSCollagen in DM is insoluble and resistant to collagenases than stromal collagenThus DM offers resistance to trauma, chemical agents, infection

4) Endothelium: Biochemical composition not properly knownEnzymes of glycolysis and kreb’s however are present

Metabolism

- for maintaining transparency and dehydration-Energy(ATP) derived from breakdown of glucose Most actively metabolizing layers are epithelium & endothelium ( former 10* more than latter)

Sources of Nutrients:

1) Oxygen Epithelium• O2 from atmosphere through tear film• also from limbal vasculature• Normal Po₂ in tears is 155 mm Hg• Tight fitting contact lens of non oxygen permeable material PMMA

(polymethyl methacrylate) interferes• O2 intake (Hypoxia)• Intracellular edema• Decrease in epithelial glycogen • Increase in lactic acid

Endothelium:• derives O₂ from aqueous• Essential nutrients (such as glucose & amino acids) pass across its

surface to supply the cellular needs of all the corneal layers

2) GlucoseGlucose metabolism is prime energy sourceAH is the main source of glucose and supplies endothelium, stroma and

epitheliumNegligible amount of glucose also comes from tear film and limbal capillaries

3) Amino acidsAmino acid, vitamins, and other nutrients supplied to cornea by AH, a lesser

amounts from tears or limbal vessels

Metabolic pathways:

-Three pathways –1. Glycolysis (Embden meyerrhof pathway) and2. Kreb’s cycleThe enzymes are present in all cells of corneaKreb’s occur less than glycolysis thus in aerobic conditions as well lactate is accumulated3. Hexose monophosphate shunt :glucose is also

metabolized by this pathwayNo net gain of ATP in thisPurpose is to produce NADPH(required for

biosynthesis of lipids by corneal epithelium)

• Glucose is mainly metabolized by Anaerobic glycolysis(70%) and about 30% enters HMP shunt• HMP converts hexoses to pentoses required for nucleic acid synthesis and

produces NADPH , a reducing agent required for fatty acid synthesis.• Glucose may enter sorbitol pathway producing sorbitol and fructose• In excess glucose, excess sorbitol is produced in lens and peripheral nerves

casuing osmotic cell damage• Aerobic conditions: pyruvate can enter kreb’s cycle• Hypoxic states: pyruvate into lactate• Lactate cannot diffuse across apical barrier and builds up intracellularly• Epithelial edema

Corneal Transparency

Anatomical factors: a) Uniform & regular arrangement of corneal epitheliumb) Arrangement of corneal lamellaec) Corneal avascularityd) Absence of lymphe) Presence of nonmyelinated nerve fibers

Physiological factors:f) Stromal swelling pressureg) Metabolic pumph) Barrier functioni) Evaporation form corneal surfacej) IOP

1. Corneal epithelium and tear film:• Tight junctions between cells account for corneal transparency and

resistance to flow of water or electrolytes • Tear film has an important role to maintain transparencyCorneal epithelial defect/ tear film abnormalities::loss of transparency

2. Arrangement of stromal lamellaeTwo theories –• Maurice (1957):

i) The transparency of the stroma is due to the lattice arrangement of collagen fibrils.

ii) Because of their small diameter and regularity of separation, back scattered light would be almost completely suppressed by destructive interference

iii) As long as fibrils are regularly arranged in a lattice separated by less than a wavelength of light the cornea will be transparent…..thus when this altered by edema …lost tranparency

ii) Goldman et al. (1968):

• Stated that Lattice arrangement is not necessary for transparency• Rather postulated that transparency is because that the fibrils

are small and do nit interfere with transmission of light• will remain transparent until they are smaller than one half

of wavelength of light

Limitations:Bot theories fail to explain occurrence of clouding of cornea

with acute raise in IOP and rapid clearing after IOP normalises

3. Corneal avascularity:• Cornea avacular• Except –small loops which invades peripheral cornea for about 1 mm

Corneal vascularization occurs in:• Various disease process • To bring defense mechanism against noxious agent• Facilitates nutrition, transport systemic drugs

Progressive vascularization thus:• Harmful • Interferes with functional process(transparency)

Chemical theory: • Avascularity is maintained by VIF (vaso- inhibitory factor)• Vascularisation of cornea occurs due to vaso-stimulatory factor(VSF) or loss of

VIF

Mechanical theory:• BVs cannot invade cornea due to its compact structure• Thus when structure is loosened ie oedema neovascularization takes place

• Some however doubt that oedema alone will lead to vascularization• Eg in Fuch’s dystrophy and aphakic bullous keratopathy it is rare for

vascularization to occur

Role of leukocytes: • vascularization occurs as a result of inflammatory process

4. Relative deturgescence state of normal cornea (corneal hydration)

Water content of cornea(80%)

Balanced by : Factors that draw water in : stromal swelling pressure and IOPFactors that act as barrier & prevent flow: corneal epitheliumFactors that draw water out: active pumping action of corneal endothelium

Disturbance in this balance:: hydration above 80% :: corneal oedema:: central thickness increases and transparency reduces

a) Stromal swelling pressure (SP): Pressure exerted by GAGs (60 mm Hg) in stroma -ve charges in GAGs repell eachother…expands the stroma…sucking in the fluid with equal negative pressure k/a imbibition pressure (IP)Net imbibition pressure = IOP-SP

b) Barrier function of epithelium and endothelium:Corneal epithelium = 2* resistance to water flow than endotheliumCorneal transparency decreased when endothelium/epithelium is damaged

c) Evaporation of water form corneal surface i.e tear film increases the osmolarity of the tear flim…draw water out of cornea.. however this loss is quickly recovered from AH and contributes less in corneal dehydration

d) IOP: when IOP > SP epithelial edema occurs ..loss of transparency

e) Endothelium • has a major role • Endothelial pumps are present which removes water from cornea continuously• Endothelial cell count however declines with age• When cell density reduces to several thousands/mm2 corneal decompensation

occurs

Hydration control by ::endothelial pumpsThese pumps are active pumps thus require ATP to function• Na/k pump: pumps Na out of cornea

• Hco3 : is generated intracellularly & via CA action CO2 is converted to hco3…transport of hco3 across the cell is energy dependent …the osmotic gradient favors movement from stroma to AH

• Na/H pump is also present

Also passive diffusions of k, cl, hco3 from cornea into AH

Na, cl, hco3 into cornea from AH

• Na+ to move into the epithelium from the tear film• Na+ extruded from via Na+/K+ ATPase

(basolateral membrane)• Na+, K+, and Cl− into the cell via

Na+/K+/2Cl− cotransporter (basolateral membrane• Cl− moves out of the cell (apical surface)• K+ : out of the cell down its concentration

gradient (basal Membrane)

• The net K+ transport creates the electrical force for Cl− diffusion across the apical side of the cell

• This Cl− movement in turn has been identified as the driving force for osmotic water transport out of the epithelial cell into the tear film

Na concentration of • AH=143meq/l• Stroma = 160meq/l• This would favour water into stroma• large portion of Na is bound by stromal ground substance • Thus osmotically active Na=134meq/l..thus,draws water out of stroma

5) Cellular factors : corneal fibroblasts(keratocytes) are source of stromal collagen and proteoglycans

• Enzyme defects can thus lead to corneal opacification

Cell turnover and wound healing in cornea:

Epithelium:• Constantly regenerated by mitotic activity in basal layer of cells• Initial response of epithelium to debridement is to migrate across stroma to close the defect• Migration of epithelial cells occurs by cytoskeletal and cell shape changes by means of

redistribution of actin myosin fibrils• Changes in actin distribution precedes changes in actin binding proteins such as fodrin and

E cadherin

• Migration is also dependant upon intracellular signaling via components such as fibronectin/fibrin, laminin and collagen peptides through surface integrins

• Fibronectin/fibrin component is important in healing when basement membrane is damaged particularly when the laminin component is lost

• Migration needs energy as glycogen is depleted during wound healing• Process also requires calcium since migration is inhibited by calmodulin

inhibitors• Cell migration is stimulated by CAMP• Following healing epithelium is reestablished with hemi desmosomes( initially hemi desmosomes disappears as cell prepares for healing)

• When BM is intact….normal epithelium with adhesion complex is formed soon after healing• When BM is damaged…adhesion complex is formed after 12 months

• When recurrent erosions occur….healing is delayed and normal epithelial adhesion is not present• After trauma discontinuity of attachment is seen for 8-12 weeks

• Adhesion of epithelium to bm and bowman’s layer is via hemidesmosomes, lamina densa and anchoring type 7 collagen fibrils• Hemidesmosomes form early in repairs (18 hrs)• Anchoring fibrils takes long time to develop• Thus recurrent erosions is common in condition where superficial stromal layer

damage have once occurred• Proteolytic enzymes implicated in repair are urokinase type plasminogen

activator and matrix metalloproteinases

• Most mitotic activity takes place at limbus• Use of Epidermal growth factor and retinoic acid to promote growth –

unsuccessful

Stroma:

Incisional wounds invoving cornea ….immediate effect is imbibition of water from tears by GAGs…opacity of corneaSeries of events occur:i. Deposition of fibrin at woundii. Rapid epithelisation of wound iii. Activation of keratocytes to divide and synthesize collagen and GAGs

Initially …there’s loss of specialization of keratocytes ….such that they revert to fibroblast like function and lay down collagen and GAGs like in other wounds (type 1 and type 3 collagens , hyaluronic acid)

Also the size of collagen fibrils are not regular

Extensive wounds ( remains as such with opacity) small wounds ( cornea attempts to restore transparency)

• Corneal curvature is due to tension in circumferential fibers

• Normal curvature wont be restored unless wound edges are apposed by surgical reconstruction

• This is basis for refractive surgery….where partial thickness wounds are left to heal in a gaping configuration

• Use of Argon-F1 and UV laser is widely used for precise incisions in stroma

• Lasik : surface of cornea is reconfigured by raising corneal flap, laser ablation of exposed stromal bed and then restoring the corneal flap without sutures

• Endothelium:

Corneal endothelium doesn’t undergo mitosis

Response to wound is to undergo “ cell slide”

With age endothelial cell number decreases, size increases

Corneal vascularization:

• Occurs when vessels from conjunctiva or deep episcleral vessels invade the periphery during healing of wound or corneal ulcers

• During Inflammatory response MMP, plasminogen activators, cytokines(IL1,IL6,IL8 and TNF) are secreted by inflammatory cells

• Initiates vascularistaion

• Vessles advance to site of injury or infection and contribute to opaque “leucoma” of healed cornea

References:

• Wolff’s anatomy of eye and orbit 8th edition• Snell’s anatomy of the eye• Internet sources• AAO external disease and cornea

Refractive surgery:

• Corneal curvature is altered to change the focusing of light rays on the cornea

• Radial keratotomy: radial incisions:- flattening of corneal curvature

• Photorefractive keratectomy : Excimer laser beam is used to ablate the cornea—change the curvature

• LASIK: cutting and raising of thin flap of cornea then exposed stroma is ablated to change the curvature of cornea

Effect of contact lens wear on corneal physiology::

Contact lens predominantly affects function of epithelium(receives O2 from tear film and glucose from limbal and aqueous vessels)

Contact lenses reduce direct availability of O2 to epithelium thus shifts metabolism from aerobic to anaerobic metabolism.

Lactate levels in cornea are doubled with contact lenses wear and CO2 production is increased

Hard contact lenses are usually made from PMMA (polymethylmethacrylate)..effect corneal function greatly

In addition to restriction of O2, hard lenses deplete glycogen stores…as inhibition of aerobic enzymes such as hexokinase reduces glucose utilization by cornea

• Soft contact lenses are made up of polymers of HEMA, silicone

• Allow extended wear of contact lens due to greater permeability of O2 and CO2

• Contact lenses have deleterious effect on epithelium:Cause ThinningReduction in hemidesmosome density and number of anchoring fibrils

In severe cases:Epithelial edema and keratopahty in the form of punctate epithelia erosions

END

PhysiologyNutrition • The endothelium plays a major role in maintaining corneal

transparency. • Essential nutrients (such as glucose and amino acids) must pass

across its surface to supply the cellular needs of all of the corneal layers; oxygen derived from the aqueous supplies the requirements of the endothelium and posterior stroma (Po2 55 mmHg).

Fluid regulation • Physiological studies have shown that this delicate monolayer of

cells is responsible for maintaining the corneal stroma free of oedema, in a state of relative deturgescence.

• This it does in two ways: 1. first it provides a barrier function to the ingress of salt and

metabolites into the stroma, which has a spontaneous tendency to take up salt and consequently, by osmosis, water;

2. second, it actively reduces the osmotic pressure of the stroma by metabolically pumping the bicarbonate ions out of the stroma and back into the aqueous humour (Fig. 7.40).

• The barrier properties of the corneal endothelium, which have recently been shown to be relatively simple, need a little explanation. On their lateral walls, posteriorly, the cells exhibit apparent tight junctions (part of a junctional complex).

• Most of the barrier function is provided by the geometry of the intercellular space, and a passive movement of ions and metabolites across the monolayer takes place through the intercellular spaces between the cells.

• Compared to most monolayers of cells, corneal endothelium is relatively 'leaky', mainly because of the shortness of the interdigitating intercellular space and the absence of a physiologically functional tight junction.

• Nevertheless, the barrier function is well balanced and matched to the activity of the outward-directed bicarbonate ion pump.

• In contrast to its solvents, the water molecules of aqueous humour and stroma pass with greater freedom directly through the cells of the monolayer ensuring that the water potential of both stromal fluid and AH is equal.

• Changes in corneal hydration are, therefore, rate-limited by the balance between the net passive diffusion of ions between the cells and into the stroma and the active bicarbonate pump which works through the cells, in the opposite direction, out of the stroma.

• In this way it can be seen that stromal hydration is regulated by a system of pump-leak of ions, chiefly under the control of the corneal endothelium. The epithelium plays a minor role in regulating corneal hydration although it has a major barrier function. The gap junctions found between corneal endothelial cells serve to facilitate cell-to-cell transport of ions and result in electrical coupling of endothelial cells.

Injury and repair• Great interest has been focused on the endothelial response to injury because of its dual

role as barrier and fluid pump of the cornea. • The pumping activity of the corneal endothelium may be inhibited experimentally by drugs

such as ouabain, and permeability of the endothelium increased by destruction of the endothelial gap junctions.

• Physical and chemical damage to the human corneal endothelium result in loss of endothelial cells, and because of the poor reparative power of human endothelium the loss in continuity of the endothelial sheet is made up by a sliding process in which neighbouring cells move over to fill the gap.

• This is accompanied by enlargement of the cells to cover the original area. Thus, after injury, the endothelial cell density falls, the cell area increases and the cell height decreases. This sliding phenomenon is not distributed equally across the whole of the corneal surface and after a localized injury will be confined to the immediate neighbourhood of the injury.

• Athough the healed endothelium may exhibit a regular hexagonal endothelial pattern, some endothelial polymorphism is common.

• Endothelial injury produces corneal oedema due to loss of the specialized junctions between endothelial cells and of the pumping function of the cells at the site of injury.

• With re-establishment of the endothelial sheet, these specialized junctions and the normal pumping and permeability characteristics of the endothelium return, with the disappearance of corneal oedema.

• The process of sliding of cells and decreased endothelial cell density is a normal ageing phenomenon, which accounts for the fall in endothelial density which occurs with age.

• Adult human endothelial cells rarely undergo cell division spontaneously. It appears that the cornea receives its full complement of cells at or about birth (about 5 000 000 cells).

• Although occasional mitotic figures are found in human corneal endothelium repair of endothelial damage is generally by slide rather than mitosis. This behaviour of human endothelial cells is of major clinical importance

STRUCTURAL PROTEINS OF THE CORNEACollagen is the major structural component of the cornea, while proteoglycan accounts for most of the 'ground substance'Collagen • .The ocular collagen types include the fibrous collagens types

I, II, III and V, non-fibrous collagen type IV and filamentous collagens types VI, VIII, IX and X. • The basal lamina of the epithelium contains type IV collagen

in its periphery. • The predominant collagen (about 90%) of the stroma is type I

and thus resembles the collagen of sclera and muscle tendon. • The proportion of other types has been variously estimated:

type II is found in the embryonic cornea; type V increases with maturation from 5 to 10% and may be the predominant collagen of Bowman's layer.

Proteoglycans• Proteoglycans constitute most of the ground substance material between the stromal fibrils, of which keratan sulphate

represents 50% and chondroitin and chondroitin sulphate the remainder. • keratan sulphate (major) + chondroitin (minor) constituent in central stroma with the constituent, while chondroitin

sulphate replaced chondroitin in peripheral cornea.• In the peripheral cornea there is an increasing proportion of dermatan sulphate and a fall in keratan sulphate.

Proteoglycan interacts with the collagen fibrils in a precise way in relation to its banding pattern (Fig. 7.43). • This banding pattern reflects the charge distribution along the amino acids of the collagen molecule, and the ordering

of the collagen fibrils in a staggered array within the fibril. X-ray diffraction and electrohistochemical staining have demonstrated dermatan sulphate to be located in the gap region as well as at several sites outside it, while keratan sulphate is located in the overlap region

• . It has been suggested that dermatan sulphate in the gap region may play a role in inhibiting calcification in normally non-calcifying tissues such as tendon and cornea and skin . Proteoglycan is found in fixed ratio to collagen in the cornea across the species. Where the collagen fibrils are narrower, the fibrils are closer and there is less proteoglycan

• Electrohistochemistry reveals that proteoglycan is attached in a ladderlike arrangement along the collagen fibrils, and also between them. Much of the stromal proteoglycan can be extracted from the cornea with weak salt solutions suggesting noncovalent attachment to collagen, therefore (although it is certain that this matrix material plays a role in resisting compressive forces on the cornea) its contribution to the tensile and shear strength of the cornea is uncertain. The proteoglycans provide the colloidal osmotic force responsible for the tendency of the cornea to swell. This is counteracted normally by the endothelial (and less epithelial) water pumps which maintain the cornea at its normal level of eturgescence and transparency. stromal oedema is accompanied by altered biosynthesis of ground substance and appearance of dermatan sulphate centrally, normally confined to the limbal region. A similar modification, with loss of keratan sulphate and increased heparan sulphate and hyaluronate, is associated with corneal scarring .

• CORNEAL TRANSPARENCY• The cornea transmits nearly 100% of the light that enters it, despite the changes in refractive index

between its elements, the collagen fibrils and ground substance of the stroma. • Maurice (1957) explained the transparency of the stroma on the basis of a lattice arrangement of the

collagen fibrils. He argued that, because of their small diameter and regularity of separation, back-scattered light would be almost completely suppressed by destructive interference. This theory was modified slightly by Goldman (Goldman and Benedek, 1967; Goldman et al., 1968) who suggested that a perfect crystalline lattice periodicity is not necessary for sufficient destructive interference to occur. Thus, if fibril separation and diameter is less than a third of the wavelength of the incident light, almost perfect transparency will ensue (Farrell et al., 1983). This is the situation which obtains in the normal cornea. Transparency is lost when this regular ordering of corneal elements is destroyed, such as in corneal scarring when new collagen fibrils are laid down which have a wide variation in separation and fibrildiameter and an irregular interweaving. In stromal Corn eal oedema, increased separation of collagen fibrils is due to formation of 'fluid lakes', and results in stromal clouding. Collagen fibril thickness probably also increases. However, in contrast to oedema of the orneal epithelium, the accumulated fluid results in an irregular epithelial surface and the irregular astigmatism produced at the air-tear interface degrades retinal image formation in a more potent manner (Miller and Benedek, 1973). In milder degrees of epithelial oedema, which may occur when wearing ill-fitting contact lenses or as a result of markedly raised intraocular pressure, the basal epithelial cells, which are regularly arranged, become separated by oedema fluid of differing refractive index to the cells themselves. This creates a diffraction grating effect so that the patient sees rainbows round white lights. This is an important symptom in subacute angle-closure glaucoma where the rise in intraocular pressure leads to epithelial oedema

Autonomic and peptidergic innervation

CORNEAL STROMA AND EPITHELIUM• In some species (e.g. rodents) this is richly supplied by sympathetic adrenergic

fibres. • Primary sensory neurons have been shown to contain substance P,

somatostatin, cholecystokinin and gastrin/bombesin-releasing peptide. • Epithelial serotonergic nerves appear to regulate inward chloride transport

which may be modulated by prejunctional dopaminergic receptors. • There is no direct evidence of parasympathetic nerve fibres in the cornea.