Corneal Biochemistry- Ocular Biochemistry

Optometry

1. Corneal
Biochemistry
Corneal
Biochemistry

2. Introduction •Corneal avascularity is maintained by soluble
vascular endothelial growth factor receptor1
(also known as sflt-1) which blocksVascular
endothelial growth factor A
•The corneal epithelium provides a barrier to
diffusion of hydrophilic molecules; however,
corneal proteoglycans confer hydrophilic
properties to the stroma
•Because of the lack of blood vessels in the
cornea, oxygen is provided to the cornea via
the precorneal tear film, or tear film , and
aqueous humor
•Glucose is metabolized in the cornea by all 3
metabolic pathways:
•hexose monophosphate (HMP) shunt
•tricarboxylic acid (TCA) cycle
•glycolysis

3. •In the epithelium and endothelium, the HMP pathway breaks down 35%–65% of the
glucose, but the keratocytes of the stroma metabolize very little glucose via this
pathway
•Human corneas possess a remarkably high level of aldehyde dehydrogenase and
transketolase
•Similar to enzyme crystallinsof the lens, both aldehyde dehydrogenase and
transketolase are thought to contribute to the optical properties of the cornea
•The following clinically relevant principles have been confirmed: The paracentral
and peripheral cornea are stiffer than the central cornea because of differing
orientation and number of collagen fibrils
•The stiffness of the cornea increases with age, apparently because of natural
collagen crosslinking

4. Epithelium
•The epithelium constitutes 5%–10% of the total corneal thickness
•Surface projections are presenton the apical surface of themostsuperficial cell layer
ofepithelium
•Itsessential biochemical components are as follows:
•Water represents 70% ofthe wet weightEpithellium
•Proteinsynthesis in epithelium isfive times higher thanthe stroma and about 2times
higher than theendothelium andDescemet's membrane
•Lipids (phospholipidsand cholesterol) aremainly present in the cellmembranes
andconstitute about 5.4% ofthe dry weight of theepithelium.
•Enzymesnecessary for glycolysis,Krebs cycle and Na, Kactivated ATPase arepresent in
high levels inthe epithelium.

5. •Normal corneal epithelium remains in a steady
state in which cell proliferation is necessary to
replace cells lost by terminal differentiation and
desquamation
•While basal cells of the central cornea proliferate
actively, basal cells at the limbus consist of a
mixture of slow-cycling stem cells and their
progeny, transientamplifying (TA) cells, which
are affected by growth factors, cytokines, and
extracellular matrix.
•During treatment of corneal wounds with
cryopreserved amniotic membrane, TA cells are
likely upregulated to enhance wound healing.
Figure - Desquamation of corneal epithelial cells.
Stem cells migrate centrally from the limbus and
give rise to transient amplifying (TA) cells and basal
epithelial cells.
Epithelium

6. Penetration of the Corneal Epithelium
•Hydrophilic molecules penetrate theepithelium poorly, but they may passthrough
tight junctions if the polar moleculehas a mass lower than 500 Da.
•Hydrophilicdrugs can also reach very high cornealpenetration levels when the
cornealepithelium is damaged or inflamed.
•Thedissociation constant (also called ionizationconstant) is likewise important
indetermining a molecule’s permeabilityacross the cornea.
•To diffuse across theepithelium, organic molecules should be inan uncharged
state.
•However, a chargedmolecule can more readily penetrate thestroma. To penetrate
the cornea and enterthe anterior chamber, therefore, an organicmolecule should
be able to dissociate atphysiologic pH and temperature (ie, withinthe stroma)

7. Bowman Layer
•The Bowman layer is immediately beneath the epithelial basal lamina and is
composed of randomly packed type I and type V collagen fibers that are 30 nm in
diameter
•The fibers are enmeshed in a matrix consisting of proteoglycans and glycoproteins
•Corneal haze, a potentially significant postoperative complication of these
procedures, is presumably due to absence of the Bowman layer and consequent
keratocyte exposure to growth factors
•In LASIK, by contrast, the Bowman layer is transected but retained; central corneal
haze is thus extremely rare after this procedure

8. Stroma
•The stroma makes up approximately 90% of the total
corneal thickness
•It contains 75 to 80% water (wet weight). Remaining
solids (20 to 25% ) include mainly extracellular
collagen, other soluble proteins,
mucopolysaccharides (chondroitin, keratan sulphate,
and dermatan sulphate) and salts.

9. •embedded in hydrated matrix of
proteoglycans, essentially constitute the
corneal stroma
•These present the typical 64-66 nm
periodicity of the collagen
•Collagen constitutes approximately 70%
of dry weight of human cornea. Type I
collagenis the predominant type,
although collagen types III, V (10-20%), VI
(15.1 % ), VII, XII andXIV have also been
found in normal adult cornea.
•The diameter of corneal collagen fibrils
(35 nm) and spacing between these fibrils
(55nm) is remarkably constant.
•The stroma is less compact posteriorly,
facilitating a deeper placement of
intrastromalring segments for
keratoconus
•Regularly arranged lamellae with uniform
diameter and separation of collagen fibre
makes cornea transparent
Collagen
fibrils
(lamellae):
•There is an inverse correlation between the
number of carbohydrate units and the fibril
diameter of collagen. The corneal collagen, like
the collagen from other structures such as skin
and tendons, has a high glycine, proline and
hydroxyproline content
•The mature collagen is a helix composed of two
alpha chains (molecular weight 80,000) and
one beta chain (molecular weight 160000)
•The corneal collagen is dissolved by proteolytic
enzymes such as collagenase, which has
important implication in corneal ulceration.
•In boiling water or acids, the corneal collagen is
converted into gelatin, which accounts for the
acid corneal burns being less serious than the
alkali burns

10. Figure - Cornea and sclera. A, Both
are composed of similar collagen
fibrils. However, fibril diameter and
fiber density are consistent
throughout the cornea, whereas in
the sclera, they are not.

11. Fig- Orientation of stromal collagen fiber lamellae. The anterior stroma is more compact than the
posterior stroma, particularly at the Bowman layer.

12. Stroma
•After collagen, proteoglycansare the second most abundant biological constituents of the
cornea; they constitute approximately 10% of the dry weight of the cornea
•Proteoglycans are a family of glycosylated proteins that contain at least one glucose amino
glycan chain covalently bonded to a protein core Glycosaminoglycans (GAG) or the so-called
acid-mucopolysaccharides represent 4 to 4.5% of the dry weight of the cornea
•Cornea contains three major GAG fractions namely: 1. Keratan sulphate (50%) 2. Chondroitin
sulphate A (25%) and 3. Chondroitin (25%), present exclusively in the cornea)
•GAGS are present in the interfibrillar space of the corneal stroma and account for the stromal
swelling pressure' (normal-60 mm Hg), i.e. its tendency to imbibe water and thus plays an
important role in the maintenance of the corneal hydration level and transparency as they have
water absorptive capacity.
•An abnormal accumulation of GAG occurs in the corneal stroma of the patients affected by the
inborn errors of GAG metabolism known as mucopolysaccharidosis

13. Stromal Matrix metalloproteinases (MMPs)
•MMPs are calcium dependent zinc containing
endoproteinase family of enzymes that
breakdown components of extracellular
matrix. In the cornea, they help maintain the
normal framework and have a crucial role in
remodelling after injury
•MMPs are secreted as proenzymes by
infiltrating inflammatory cells or by cells
resident in the tissue
•They are then activated by cleavage of a
peptide from their N-terminal end
•All MMPs require a metal cofactor
•The MMPs of cornea have different substrates:
•MMP-1 (collagenase-1) is active against collagen
types I, II, and III. MMP-2 (gelatinase A) andMMP-9
(gelatinase B) are active against collagen types IV, V,
and VII as well as gelatins andfibronectin.
•MMP-3 (stromelysin1) breaks down protein glycans
and fibronectin MMPS 7, 8, 9, 11 are othersubstrates
Note.
•Only MMP-2 has been detected in normal cornea,
the other MMPs mentioned above are foundin the
cornea only after injury.
•MMPs 1,2 and 3 are products of stromal cells,
whereas MMP-9 is produced by the
cornealepithelium which is most importantly
involved in the corneal inflammation

14. Stromal swelling
pressure
•SP, 60 mmHg
•Pressure exerted by
glycosaminoglycans(GAGs) of the
corneal stroma which act like a sponge
•Electrostatic repulsion of the anionic
charges on the GAG molecule expands
the tissue, sucking in the fluid with equal
but negative pressure called, imbibition
pressure (IP) In vitro, IP-SP In vivo, IP is
reduced by values equivalent to IOP. i.e.
IP=IOP-SP i.e. IP=17-60=-43mmHg.
•Negative imbibition pressure draws out
water from stroma.

15. Descemet
Membrane and
Endothelium
The Descemet membrane is a specialized
basement membrane, 10–12 μm thick,
between the corneal endothelium and the
posterior stroma
It is secreted by endothelium and comprises
an anterior banded layer and a posterior
nonbanded layer
The latter is secreted throughout life, which is
the reason why the Descemet membrane is 3–
4 times thicker in adulthood than at birth
Type IV is the most abundant collagen in the
Descemet membrane

16. Endothelium
•The corneal endothelium, located posterior to the Descemet membrane, is a
monolayer of hexagonal cells with a diameter of 20 μm
•The number of endothelial cells is higher in the periphery and decreases with age,
with concomitant spreading and thinning of the remaining cells
•Adjacent endothelial cells interdigitate in a complex way and form a variety of tight
junctions, serving as a barrier to aqueous humor penetration, but desmosomes are
never observed between normal cells
•Approximately 20–30 short microvilli per cell extend from the apical plasma
membrane into the aqueous humor

17. Endothelium
•The major non-aqueous constituents of the stroma are collagen fibrils and proteoglycans
•The collagen fibrils are made of a mixture of type 1 I and type V collagens These molecules are tilted by
about 15 degrees to the fibril axis, and because of this, the axial periodicity of the fibrils is reduced to 65
nm (in tendons, the periodicity is 67 nm) The diameter of the fibrils is remarkably uniform and varies
from species to species. In humans, it is about 31 nm
•Proteoglycans are made of a small protein core to which one or more glycosaminoglycan (GAG) chains
are attached.
•The endothelium functions both as a permeability barrier between the aqueous humor and the corneal
stroma and as a pump to maintain the cornea in a dehydrated state by generating negative hydrostatic
pressure, which also serves to hold free corneal flaps in place
•Endothelium utilizes temperature-dependent Na+, K+ -ATPase to maintain the hydration of the stroma at
78% and sustain corneal clarity
•If the endothelium is injured, healing occurs mainly via migration, rearrangement, and enlargement of
the residual cells

18. METABOLISM OF CORNEA
The corneal epithelium plays several roles in the process of image formation
The scattering of light by the epithelial cells decreases the clarity of image
The common cause of intraepithelial cell edema is epithelial hypoxia resulting from contact lens over
wear or intercellular edema may result from high IOP, as is seen in acute angle closure glaucoma
Barrier function
Demonstraterefractive function
Responseto wound and smoothing tendency

19. Barrier Punction
It is the first line of barrier between the external environment and the corneal stroma.
It prevents movement of fluid from tear to stroma and is useful for maintenance of epithelial
barrier.
The maintenance is done by mitosis of basal cells and migration of new basal cells from the
limbal stern cells. So, the corneal epithelium is maintained by a balance among the process
of centripetal cell migration (120pm/wk), mitosis, and shedding of superficial cells.
The XYZ hypothesis of corneal epithelial maintenance by Thoft and Friend is X+Y=Z. The
limbal epithelium has the highest mitotic rate followed by peripheral cells and central cells
has the lowest mitotic mte.

20. Refractive Function Cornea
Through its interaction with the tear film, it forms a smooth
refractive function and uniform thickness

21. Response to
Wound
Response to wound initiates process of migration
centripetally across the basement membrane to cover the
aberrated area and trigger a mechanism which is not known.
The migration starts about 5 hrs after injury at the rate of 60-
80 mm/hr until wound closes.
There is mitosis and migration of cells.
Hemidesmosoines disappear from basal cells during lag
phase. lncrease in vinculin synthesis occur during cell
migration.
Along with this there is synthesis of cell surface glycoproteins
and glycolipids during wound healing.

22. Smoothing Tendency
In health and in disease condition the epithelium has a strong
tendency to smoothen the underlying irregularities, e.g.,
epithelial facet by epithelial plug filling
This smoothing tendency is also active in corneas after excimer
photoablation therapy. There is a tendency of the epithelium to
bring the underlying area back to mitigate the refractive effect
of the ablation.

23. Electrophysiology
and Ion
Transport of
Cornea
The trans-epithelial
potential is 25-35 mV
Metabolism and
Respiration of Cornea
The cornea requires
energy lor he maintenance
of its transparency

24. Metabolic Pathways
1) 88 per cent of glucose is utilized through glycolysis/ Embden
Meyerhof Parnas Pathway
2) Only 12 per cent of glucose is utilized by TCA cycle (Kreb's
Cycle).
3) HMP shunt is also important for production of NADPH and
ribose.

25. Fig - Principal pathways of glucose metabolism in the corneal epithelium.
The overall glycolytic and oxidative pathways are similar to those found in
non-ocular tissues and in other ocular tissues such as the retina.
In the epithelium and endothelium, the HMPpathway
breaks down 35%–65% of theglucose, but the
keratocytes of the stromametabolize very little glucose
via thispathway. The keratocytes lack 6-
phosphogluconate dehydrogenase, animportant
enzyme in the HMP pathway.
Pyruvic acid, the end product of glycolysis, isconverted
either to carbon dioxide andwater (via the TCA cycle
under aerobicconditions) or to lactic acid
(underanaerobic conditions).
Production of lactic acid increases inconditions of
oxygen deprivation, as in thecase of tight-fitting contact
lenses with lowoxygen permeability.
Accumulation of lactic acid in the cornea
hasdetrimental consequences for vision, suchas
edema (due to an increase in an osmoticsolute load)
or stromal acidosis, which canchange endothelial
morphology andfunction.

26. Corneal -Hydration
• Normal cornea maintains itself in a state
of relative dehydration (80% water content)
which is essential for corneal transparency
It is kept constant by
1. Factors which draw water in the cornea,
like -Stromal swelling pressure (SP) -
Intraocular pressure (IOP)
2. Factors which prevent flow of water in
the cornea -Mechanical barrier function of
epithelium & endothelium
3. Factors which draw water out of cornea-
active pumping action of endothelium

27. Hydration control by active pump
mechanism
a) Na+/K+ ATPase pump system:
Endothelium is more active than &epithelium,
Pumps are located in basolateral membrane of endothelial cell.
Stromal transparency develops 13-20 days after birth due to greatest increase in
pump sites/cell
Enzyme "Na+/K+ activated ATPase" mediate pump causes extrusion of the Na+ &
water from the stroma and thus maintain corneal transparency Corneal hydration
depend on extent to which endothelial barrier and pump function can be
reestablished

28. Hydration control by active pump
mechanism
b) Bicarbonate dependent ATPase present in endothelium are also reported to have
role in fluid /ion balance in the cornea
c) Carbonic anhydrase enzyme catalyzes the conversion of CO2 and water into
HCO3-and H+ thus provides important source for HCO3 -for endothelial pump.
d) Na+/H+ pump has also been postulated
e) Intraocular pressure (IOP):
As we know, IP IOP-SP, i.e. 17-60=-43 mmHg, i.e. I P isanegative pressure. When IOP
exceeds SP, i.e. when IP becomes positive,cornealoedema results. It can occur when
there is -high IOP and normal SP, as inacuteglaucoma, -Normal IOP and low SP, as in
endothelial dystrophy

29. Thank You!!!