Importance of Corneal Physiology:

Basis of understanding corneal physiology: to address the physiologic importance of corneal epithelial, endothelial barrier and metabolic pump function.

Corneal Epithelium Functions:

To form a Barrier between the environment and the stroma of the cornea.Barrier formed as the cells move superficially to the surface of the

cornea ,differentiating until the superficial cells form two layers of the flattened cells encircled by the tight junction, which serves as a high resistance, semi permeable membrane.

Barrier functions to prevent the movement of the fluid from tears to the stroma .

To form a smooth refractive surface on the corneaProtects the cornea and the intraocular structures from infection

by pathogens smoothes the surface of the cornea, increases its ability to

become wet by aqueous, thereby forms smooth optical surface required for clear vision.

Contd......

Maintenance of the Epithelium:Maintained by a balance among: (X,Y,Z Hypothesis)

Process of cell migration: Migration of new basal cells into cornea from limbus. Migrate centripetally at about 120 μm/wk originate from stem cells in limbal epithelium

Mitosis: Occurs only in basal cell layer Daughter cells move upward from basal layer, differentiating into

wing cells and finally into superficial cells

Shedding of superficial cells: Corneal epithelium: Stratified Squamous epithelium from

which terminally differentiated, superficial cells continuously shed

Epithelium turn over completely every week

WOUND HEALINGEpithelium normally responds to wounding by initiation of the

process of migration across the basement membrane to cover the abraded area and reestablish the barrier.

cell migration occurs about 5 hrs after wounding – cells traveling at a constant rate of 60-80um/hr till wound closes.

During lag phase – cellular organization occurs –hemidesmosomes disappear from basal cells and biochemical processes prepare the cell for healing.

The leading edge of the migrating cells is only one layered but the wound is actually covered by multilayered sheet made of basal and squamous cells.

During corneal healing from conjunctiva, goblet cells also migrate onto the cornea –following wound closure by biochemical transduction, loss of goblet cells occurs.

Large number of reorganization and synthetic changes involved in corneal epithelial cell migration.

Migration needs energy as glycogen is depleted from the cell at the wound edge during healing .

Process also requires calcium since the migration is inhibited by calmodulin inhibitors

Contd...

cell migration is stimulated by cAMP, increased in migrating cells

increase in protein synthesis during wound healing

a specific increase in 110-kD cytoplasmic protein –Vinculin, promotes the interaction of actin with adhesion proteins(talin and integrin)

Following the healing, epithelium is re-established by the formation of hemidesmosomes

When a corneal abrasion is limited to the epithelium and the basement membrane is not damaged a normal epithelium with adhesion complex is formed soon after healing

In wounds with basement membrane damage adhesion complex is formed after 12months

Simple epithelial abrasion heals quickly without any complications

In lattice dystrophy, amyloid deposition between epithelium and bowman's layer-disrupting epithelial attachment complex.

Human cornea generates 25- 35 mV transepithelial potential

This high voltage is consistent with low ionic conductance of the apical epithelial cell membrane and high resistance of the tight junctions of paracellular pathways.

The transcellular and the paracellular potential are dependent on the existence of the apical tight junction.

The potential is due to ionic gradient set up by the epithelial transport of sodium and the chloride ions.

50% of the short circuit current across the corneal epithelium carried by chloride ions moving through apical membrane channel into the tear.

Sodium ions are pumped towards the stroma from the cell by the Na –K ATPase pump in the basolateral membrane of the cells- maintains the inward sodium gradient.

Sodium –chloride cotransporter, present at the basolateral membrane facilitate the influx of sodium down the concentration gradient carrying with it chloride ions.

Chloride ions then diffuse through the apical membrane

Corneal epithelium also contains the Na- H exchanger and a lactate –H co transporter- helps to maintain the intracellular pH by extrusion of the lactate and H ions.

Cyclic AMP also increases the conductance of apical chloride channels, stimulating chloride transport.

These systems are working homeostatically in the corneal epithelium to maintain the epithelial barrier.

....contd

Metabolic Pathways

Corneal epithelium uses primarily the glucose and the glycogen for the energy production.

Glucose reaches the cell by the diffusion from the aqueous humor

Corneal epithelial cells store high level of glycogen

Glycogen is rapidly depleted under stress as in hard contact lens wear or trauma

Glucose is primarily metabolised by the anaerobic glycolysis (Embden Meyerhof pathway) and about 35% of the glucose enters the HMP shunt.

HMP shunt converts hexoses to pentoses required for the nucleic acid synthesis and produces NADPH, a high energy reducing agent required for the fatty acid synthesis, both of which are required by the tissues with high mitotic index e.g. corneal epithelium.

Contd....Glucose may also enter the sorbitol pathway producing –sorbitol and

fructose

In the presence of excess glucose, excess sorbitol is produced in the lens and peripheral nerves causing osmotic cell damage.

Contd.

Corneal epithelium receives oxygen directly from the atmosphere under open eye condition with the partial oxygen pressure in tear film being 155mm Hg.

When the lids are closed oxygen pressure drops to 55 mm Hg- apparently adequate to maintain the epithelium

Slight epithelial oedema occurs during sleep

The cornea consumes about 3.5 uL O2 /cm2 /hr

Under aerobic condition pyruvate from the glycolysis can enter the kreb’s cycle(TCA Cycle)

Under hypoxic condition pyruvate is converted to lactate which is transported from cells to maintain intracellular pH at 7.3 – 7.4

Lactate cannot diffuse across the apical barrier and builds up in the intracellular space of the epithelium and stroma

•Clinically, corneal stroma appears homogenous and transparent but there is difference between anterior and posterior corneal stroma.

•The anterior stroma has less water (3.04gm H20/gm dry weight) compared to the posterior stroma (3.85gm H20/gm dry weight) – the difference being atmospheric drying of corneal epithelium

Anterior stroma has less glucose than the posterior

More of the dermatan sulphate is present anteriorly -has less water sorptive property and greater water retentive property

While keratan sulphate is located posteriorly –has greater water sorptive property with little retentive property

So, clinically most oedema occurs in posterior stroma

A greater degree of corneal swelling occurs when endothelial barrier is removed compared to epithelial barrier.

Once the barrier or metabolic pump function is lost, corneal stroma swells because of the hypertonicity of the stromal collagen, salts and proteoglycans compared to tear and aqueous humour.

corneal stroma swells on endothelial disruption as IOP of 15 mm of Hg is unopposed and aqueous water will diffuse into the stroma.

Loss of either barrier and uptake of water by stroma, loss of proteoglycans( Dermatan and keratan sulphate)

The relative resistance to the nonedematous epithelium,stroma, endothelium is 2000:1:10 thereby restricting the electrolytes to the stromal compartment

Stroma has the property to imbibe water – reflects the water imbibing property of the proteoglycans.

Stromal edema occurs in the ground substance leads to increased spacing of the collagen fibrils

Contd....

Contd...

In cornea of normal thickness, swelling pressure is 60mmHg and is inversely related to the corneal thickness

The swelling pressure of the stroma relates to the electrostatic repulsion of the negatively charged glycosaminoglycans (keratan sulphate, dermatan sulphate, chondroitin sulphate)

The actual concentration of the Na+ and K+ is higher in the stroma than in the aqueous

Contd....The ionic activity is low in the stroma than aqueous

reflecting cationic binding sites on the stromal GAG–thus favoring the movement of water from stroma to the aqueous.

The ionic activity which determines the osmotic and diffusional gradients for Na is less in the stroma than the aqueous reflecting cationic binding by the anionic sites.

This binding decreases the effective osmolarity of the stroma thus favours water movement from the stroma to the aqueous.

Contd....Water evaporates from the corneal surface at a rate of

2.5uL/cm2/hour.

Evaporation accounts for 5% thinning of the cornea during the day time compared with the thickness measured at morning after night sleep.

The normal cornea maintains a constant thickness in the presence of IOP of 50mm Hg because the stromal swelling pressure is also in the same range.

Contd...

- Negatively charged stromal GAG tend to repel each other producing swelling pressure(SP).

- The relation of swelling pressure of stroma to IOP is IP(imbibition pressure of corneal stroma) =IOP- SP

- As stromal pressure decreases precipitiously by increase in corneal thickness, mild corneal edema combined with increase in IOP leads to high imbibitions pressure and subsequent microbullous formation and epithelial edema.

In eyes with increased IOP or with abnormal endothelial function there is epithelial edema and increased stromal thickness.

Arcus senilis consists of cholesterol, cholesterol esters, phospholipids and triglycerides-Lipids found extracellularly in stroma in hour glass pattern.

Limbal vessels – increasingly permeable with age-LDL pass into cornea and carry lipids into the eye.

Dellen-areas of localized thinning of cornea due to reversible H20 loss from stroma.

Involves :

Resynthesis and crosslinking of the collagenAlteration in the proteoglycan synthesisGradual wound remodelling and restoration of the tensile strength.

-Within 2-6 hours of penetrating injury, PMN cells appear around areas of necrosis, start proteolytic debridement of necrotic debris.

-Within 3 days, keratocytes reach the wound edge,line up parallel with wound margins and secrete collagens mainly type II and GAG (keratan sulphate).

Background

First PMN cells reaching stromal wound come from limbal vasculature via the tear film, others by migration through the stroma.

By the end of 1st week, fibroblasts and PMN invade fibrin plug.

Tensile strength of wound slowly improves with increasing deposition of collagen.

By the end of 8 week, numerous fibroblastic cells.

Wound strength continues to increase 3-6 months.

Contd...

Scar tissue becomes more compact and blends into the adjacent stroma.

Tensile strength in corneal wounds increase gradually upto 4th postoperative year.

Incision in cornea far from limbus heal more slowly than the peripheral or in chronically inflamed cornea with the neovascularisation.

Maurice Theory(1957) According to this theory, The stromal collagen fibrils are of equal diameter(275-350 Å)

and are equally distant from each other, arranged as a lattice with the inter fibrillar spacing less than a wave lenght of light (4000-7000 Å)

The tangential rows of fibrils act as diffraction gratings, may result in destructive interference of all scattered light and constructive interference of rays in the line of incident rays.

Contd... - Hence rays traveling in this plane would be enhanced while

those diffracted are cancelled out, thus giving the transparency. Goldman and Benedek'sTheory(1967) If fibril separation and diameter is less than a third of the

wavelength of the incident light,almost perfect transparency will insue. The stromal fibrils are small in relation to the light and do not interfere with the light transmission ,unless they are larger than one half a wave length of light.

Corneal TransparencyCorneal Transparency

Anatomical factors:uniform regular arrangement of the epithelium, absence of blood vessels and lymph vessels and nonmyelinated fibers, packed stromal lamellae of uniform size

Physiological factors:Stromal swelling pressureMetabolic pump Barrier functionEvaporation from the corneal surface Intraocular pressure

ENDOTHELIAL PHYSIOLOGY:

Endothelium functions as a permeability barrier that restricts the movement of water and solutes into the hydrophilic stroma.

An intact monolayer of endothelial cell –prerequisite to maintain corneal transparency.

If integrity breached, corneal oedema develops.

Corneal endothelial barrier is physiologically classified as "leaky".

Resistance across the endothelium is more than epithelium, water and solutes can traverse the endothelial layer more readily.

In a normal cornea, aqueous humour cross the endothelium and enters the stroma at a slow but constant rate.

This constant leak of aqueous provides the principal source of glucose, aminoacids and other nutrients for the avascular cornea.

Permeability of endothelial barrier also due to apical tight junction and gap junctions.

In spite of the stromal swelling pressure, it does not swell due to two factors :1. barrier function – incomplete compared to the epithelial

barrier2. endothelial pump function

When the endothelium is disrupted the cornea swells at 127μm/hr, while with disruption of the metabolic pump it is 33 μm/hr and this swelling represents the movement of fluid and solutes from the aqueous to the stroma through the incomplete barrier.

Contd....

But the constant leak of the fluid to the stroma would lead to the swelling of the stroma and loss of transparency

Volume of fluid leaking into stroma is equal to volume of fluid actively removed from the stroma by the endothelium- pump leak hypothesis.

Population of endothelial cells declines constantly throughout the life.

Despite the loss, normal thickness and transparency are maintained.

When cell density declines to several hundreds cells/mm2, corneal decompensation occurs.

At birth, endothelial density is about 6000 cells/mm2, falls by about 26% in 1st year.

Cell density falls to 2500-3000 cells/mm2 in the adult cornea, for a total of about 400,000 cells.

0.6%/year decrease of endothelial cell density

Rate of loss slows and stabilizes around middle age.

After injury of any kind, damaged cells are replaced by spread of cells from adjacent zones, cells increasing in area.

No mitotic activity in endothelium after birth.

corneal function can be maintained at 300-600 cells/mm2.

If endothelial cell count<2000 cells/mm2 - not useful for corneal transplant.

Fuch's endothelial dystrophy-due to abnormality in the final phases of differentiation of endothelium from neural crest cells.

Endothelial PhysiologyRelation to Temperature

cornea swells in cold temperature and returns to normal on warm normal temperature.- known as temperature reversal.

Maintenance of the corneal hydration is metabolic, energy dependent process.

No active mechanism that transports water is present thus no fluid pump exists.

Water moves osmotically down the gradient set up by the active transport of the ions and amino acids through the metabolic pump.

Temperature reversal effect still occurs in the absence of epithelium.

Endothelial ion TransportEndothelial ion Transport Several ion transport system exist in the corneal endothelium. Mechanism by which endothelium removes fluid from stroma

referred as endothelial pump.

1. Na- K ATPase - Essential component of endothelial pump function. - Integral membrane protein located in the basolateral aspect of

endothelium – its action vital in the maintenance of corneal hydration.-Central to all the system as maintains the sodium gradient required for the Na -H exchange thereby promoting bicarbonate production.

2. Na –H pump –

Located at the basolateral aspect .Moves sodium into the cell and water outward. Acidifies the ECF by increasing the level of CO2 that

diffuses into the cell. Carbonic anhydrase converts CO2 to hydrogen ion and bicarbonate which are transported out of the cell by basolateral Na-H pump and apical bicarbonate transport respectively.

Contd....

Contd....

3. Bicarbonate

Is essential for the maintenance of the corneal thickness .

The bicarbonate transported by the endothelium is generated intracellularly via the action of carbonic anhydrase.

CO2 diffuses into the cell from the extra cellular space combines with the water in presence of the carbonic anhydrase . The carbonic anhydrase dissociates into H and bicarbonate ions.

Contd.....The transport of bicarbonate across the cell is energy

dependent- for which there is existence of the bicarbonate –sodium transporter moving these ions in the 2:1 ratio.

The osmotic gradient favours the movement of water from stroma to the aqueous

The sodium concentration of the aqueous is 143mEq/l that of the stroma is 160meq/l which favour the inward movement of water from the aqueous

A large portion of these sodium is bound by the stromal ground substance leaving an osmotically active sodium concentration of 134mEq/l in the stroma thus drawing water out of the cornea

The bulk of water movement occurs through the cells through the glucose channels.

A pH range of 6.8 –8.2 must be maintained to prevent corneal edema. Exposure of cells to outside this range – cells swell ,become vacuolated, functional breakdown occurs, corneal thickness increases - is a result of leak as well as the decreased ion and water transport.

This is an important factor to consider in use of the irrigating solution as it may disrupt the pH and cause corneal edema.

The osmotic tolerance of the endothelium is 200-400mOsmol/kg with optimum of 304mOsmol/kg.

Endothelial wound Healing

Little or no mitosis occurs in the human corneal endothelium

In injury of any kind, defect covered by spreading of the cells from adjacent area to the wound.

within hours, adjoining endothelial cells attenuate with extensive cytoplasmic processes and migrate into the wound.

Depending upon the size of the wound, the entire defect can be recovered within 1 or more week.

Regenerating endothelium deposits new layer of DM.

May take months to years.

When a single cell is lost , the cells in the surrounding spread to fill the defect –over the time marked cell enlargement occurs-typical of aged cornea.

In larger defect, extensive cell migration occurs at 80- 100uM/day initially elongating towards the centre and finally close the wound, followed by remodelling.

Factors responsible for the cell spread are –actin stress factors EGF, extracellular matrix and glycoprotein.

Corneal vasculature

Normally avascular and transparent

Avascularity is due to compactness of normal corneal tissue

When cornea swells, becomes less compact, spaces are opened up through which vessels can grow

Vascularization is a defensive mechanism by which the cornea tries to eliminate toxic influences.

Is a sequelae to:

1. Various conditions –ocular surface disease , staphyloccocal hypersensitivity, immunologic disturbance, cicatricial pemphigoid, collagen vascular disease.

2. Inflammation 3. Hypoxia 4. Dystrophic condition

Contd....Pathophysiologya result of sprouting of limbal vessels

New capillaries arise from the perilimbal capillaries and parent venules by focal degradation of the venular basement membrane followed by movement of endothelial cell toward the angiogenic stimulus .

Vascular endothelial migration and proliferation occur in a pattern suggestive of directed growth towards neovascular stimulus, caused by the substance diffusing from the point of injury.

The migrating vascular endothelial cells elongate and form a solid sprout which later develops lumen.

Specific angiogenic factors leading to corneal neovascularisation:

acidic fibroblast factor basic fibroblast factortransforming growth factor.

Corneal Pharmacology

The volume of normal adult tear film 7-9μL and the maximum amount cul de sac can maintain : 25 to 30μL

Volume of a drop is approx. 40 μL .

Most of the medication is immediately lost to eye lashes and runs out of the eye and the remainder is diluted by tear film to approx. 25%.

Both volume and concentration of drop are reduced.

Smaller, more concentrated drops are more effective but increased cost.

Normally concentration is reduced to 90% by tearing alone in 20 min.

If irritating drop, reflex tearing causes more rapid dilution.

Ointment is retained in cul de sac, gradually melts, releasing the drug into the tears, purpose is to increase the time the drug is present in tears

After topical administration most of the drug enters the stroma, aqueous by corneal penetration

Drug penetrating to conjunctiva is carried away by blood vessels.

The corneal epithelium provides an initial barrier due to the tight junction

Epithelium composed of lipids so non polar substance penetrate readily

Stroma contain mainly H2O so polar group pass more readily

Contd....

Drugs must pass through both barriers, those soluble in both lipids and water exhibit best penetration.

Some preservatives present in drug e.g. benzalkonium chloride impair the integrity of epithelial barrier and increase penetration.

Corneal Nerves

Sensitivity present in the cornea are conducted to the CNS by the Ophthalmic division of the trigeminal nerve.

sensation of pain is well developed in the cornea.

sensitivity of the cornea increases from the periphery to the center

The sensitivity corresponds to the distribution of the nerve as there is free nerve endings in the center.

Horizontal meridian is more sensitive than the vertical and the temporal more than the nasal.

Pain of corneal irritation is characterized by foreign body sensation

Photophobia or pain arising from the exposure to light is due to the referred pain from the close association of the fifth nerve to the optic nerve.

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