Physio and Biochem of Cornea Flashcards
Functions of the cornea
major refractive surface of the eye
biodefense system
protect intraocular contencts
Functions of the cornea require what characteristics?
smooth anterior surface - glycocalyx and mucin layer of tears
transparency - arrangement of collagen in stroma, relative dehydration
normal thickness
intact epithelium and endothelium - cell jxn
normal corneal contour - collagen
epithelium cell replacement equilibrium
cell division, cell maturation, and cell death
epithelium cell maintenance
complete turnover 7-10 days (involution, apoptosis, desquamation)
limbal stem cells help replenish/divide/provide new basal cells for epithelium of cornea
limbal stem cells
located in basal cell layer of limbal epithelium
lifetime capacity for self-renewal
palisades of vogt - niche microenvironment that house the limbal stem cells
-less susceptible to UV damage
stem cells have asymmetric division
transformation of daughter cells and then differentiate to:
transient amplifying cells (basal epithelial cell)
- centripetal migration
- finite rounds of mitosis
transient amplifying cells differentiate to 1) wing cell (postmitotic) 2) superficial squamous cell (differentiated)
epithelium smooth surface by:
glycocalyx: glycoprotein, glycolipid, carbohydrate molecules, and MAM embedded in plasma membrane of superficial epithelial cells
MAMs (membrane anchored mucins): produced by superficial epithelial cells that are anchored to apical surface
optically important for smooth surface -> allows hydrophilic spreading of tear film
epithelium barrier
zona occludens - tight jxns
restrict paracellular mvmt of ion and water soluble molecules
more resistant than endothelium
bowman’s layer - normal maintanence and fxn
anchoring fibrils: type 7 collagen
stabilizes association between surface epithelium, BM and underlying stroma
anterior limiting lamina -> provides shear strength and barrier
stroma - normal maintenance and fxn
transparency
mechanical strength: arrangement of collagen bundles to tighter cohesive strength
corneal curvature: refraction - rigidity maintain corneal curvature
cells of the stroma
keratocytes: quiescent cell (fibroblasts), gap jxn - fxnl synctium, synthasize procollagen and corneal crystallins (for transparency)
also in stroma: lymphocytes, neutrophils, plasma cells, macrophages
Stroma - Collagen
anterior 1/3
thin, less regular obliquely arrange lamellae
interweave and rigidity contribute to corneal curvature
resist change to stromal hydration
shear resistance
stroma - collagen
posterior 2/3
thicker, arranged at approximate right angles
can develop folds with increased hydration
contributes to tensile trength
stroma collagen: role in transparency
fibrils in parallel orientation lamellae extend limbus to limbus
fibrils small diameter (23nm)
close packing and uniform distance between fibrils (41.4 +- 0.5 nm) -> ensures min. light scattering
lamella ensure uniform distribution of tensile strength radially in all directions
each fibril surrounded by proteoglycans -> GAGs neg charged
stroma - non collagenous proteins: proteoglycans
proteoglycans - ground substance
-core protein with GAG side chain negatively charged (chondroitin/dermatan sulfate, keratan sulfate, heparan sulfate)
- GAGs contribute to tendency of stroma to swell
- anionic group repel
- colloid osmotic pressure (cation pull in water)
fxn:
1) provide tissue volume
2) surround collagen fibrils to create uniform spacing between fibrils
3) act as pressure extering polyelectrolyte gel -> contribute viscoelastic properties to cornea
- ocular response analyzer give corneal hysteresis which shows viscoelstic tendencies
Stroma -non collagenous proteins: keratocytes
Corneal crystallins: aldehyde dehydrogenase (ALDH3A1)
contribute to cellular transparency
-reduce light scattering in cornea
also antioxidant to prevent UV-induced damage
Stroma content - relative dehydration
water 78%
collagen 15%
other 7%
Endothelium normal maintenance and fxn
single layer
metabolically active
focal or leaky tight jxn to allow paracellular transport (of nutrients)
help maintain relatively dehydrated state and transparency
synthesize descemet’s membrain
endothelial pump
pump-leak hypothesis:
rate of leakage of water and solutes into stroma is balanced by transport of ions out of the stroma -> osmotic gradient generated
equilibrium maintained if 1) stroma remains relatively dehydrated and 2)cornea remains transparent
Corneal fxn summary
1) major refractive surface of eye
2) biodefense - intact barriers
3) protect intraocular contents
epithelium - smooth optical surface, tight jxn, cell replacement
bowman’s layer - barrier, resists shearing damage
stroma - transmits light, transparent, contour, resists shearing damage and provides tensile strength,
endothelium - regulates hydration and thickness, provides nutrients
Corneal transparency requirements
smooth optical surface
uniform and regular arrangement of epithelium -homogeneity of refractive index
arrangement of collagen fibrils (small diameter, uniform diameter and spacing)
corneal crystallins
corneal deturgescence (regulate hydration, relative dehydration)
absence of vasculature
Corneal transparency theory
Benedek’s theory
collagen fibrils are: uniformly small diameter (22.5 - 35 nm), closely spaced together (45-55 nm), quasi-random arrangement
when distance between scattering structures is small, less than 1/2 wavelength of light (200nm) -> media transparent
stromal hydration (swelling) and transparency
cornea transparent when relatively dehydrated (78% water)
GAGs surround collagen fibrils
- adv: transparency
- risk: swelling due to neg. charge attract Na and absorb water
can swell up to 98% water
factors that contribute to stromal hydration (swelling)
Swelling pressure (SP) - stroma wants to swell, electrostatic repulsion between neg. charges of GAGS creates force to expand tissue and draw fluid in - typically 55 mmHg at normal
imbibition pressure (IP) - neg. pressure drawing fluid into cornea normally ~40 mmHg
intraocular pressure (IOP) - if withing normal range, no effect
- glaucoma w/ elevated IOP have higher incidence of corneal edema
- endothelial dysfxn can cause swelling
damage to endothelial layer results in more swelling due to IOP than epithelium
Necessary prerequisites to maintain relatively dehydrated state
1) tear film hypertonicity
-open eyes - hypertonic tears
-closed eyes - isotonic tears
(waking up -> symptomatic)
2) intact cellular barriers
3) active metabolic pumps
- epithelial pump: electrical imbalance, osmotic gradient, water follows Cl out to tears through aquaporins
- endothelial pump: Na/K ATPase pump, bicarbonate-dependent ATPase, aquaporin 1
- -water follows net transport of Na and HCO3 from stroma to aqueous by active transport
- -establish osmotic gradient
corneal transparency summary
collagen fibrils (uniformly small diameter, closely spaced, quasi-random arrangement)
crystallins - cells
relative dehydration - hypertonic tears, intact cell barriers - aquaporins, metabolic pumps -endothelium (na and HCO3) and epitheliam (Cl)
Clinical Edema
edema is swelling
minor if <5%:
- little effect on refractive, transparency, and mechanical fxn
- increases during sleep due to reduced oxygen levels and decreased evaporative loss
major if >5%: can cause light scattering and loss of transparency
corneal swelling - epithelia
extracellular - water vacuoles form b/n cells
intracellular - fluid in cell, scatters light
decreases VA, causes halos and pain
corneal swelling - stroma
GAGs retain water - collagen fibrils do not change diameter
causes irregular spacing -> scattered light and loss of transparency
accumulates in post 2/3 vs. ant 1/3 -> swells in posterior direction
-folds in decemet’s membrane
general metabolism
energy required: transparency, mitosis, cell migration
unique environment: temperature, avascularity
source of nutrients
tears: oxygen to epithelium (155 open, 55 mmHg closed)
aqueous: oxygen (40 mmHg), glucose (stored as glycogen in basal epithelium), amino acids, vitamins
limbal blood supply (not much): negligible oxygen and glucose, typically only peripheral cornea
carbohydrate metabolism
glycolysis:
epithelium takes most glucose from stroma (80% metabolized to pyruvate and converted to lactic acid; metabolism by PPS -> impt for cell turnover and generation of reducing agents)
endothelium metabolically active -> lactate can accumulate
hypoxic condition
glycolysis leading to accumulation of lactate
protein metabolism
epithelium has highest rate of protein synthesis
proteins of cornea:
1) epithelium - keratin, actin
2) stroma - collagen, proteoglycans
3) ECM - laminin, fibronectin
4) glutathione
Vitamin A
maintenance of ocular surface
retinoic acid - active maintains non-kertinaized epithelium
vitamin a deficiency
nyctalopia (night blindness) - initial and most common ocular manifestation of vit. A deficiency
squamous metaplasia - kertinization of epithelium
mucin formaction affected
xerophtalmia (blindness in children)
corneal wound healing - what are important?
cytokines, growth factors, and proteases together regulate the healing process involving cell death, migration, proliferation, differentiation, and ECM remodeling
basement membrane controls bidirectional movement of cytokines and growth factors b/n epithelium and stroma
sensory nerve endings release neuropeptides -> essential for corneal homeostasis and wound healing
Wound healing - epithelium
equilibrium of cell division, maturation, and death
wound healing - epithelium phase 1
latency (4-6) hours
transformation to facilitate migration:
- metabolic activity increased and cell structure reorganized
- damaged epithelial shed after apoptosis -> release cytokines (IL-1 and TNF-alpha) causing keratocyte apoptosis in superficial stroma and immune cell infiltration to remove debris
mitosis CEASED for 24 hours
cell jxn lost at edge of wound -> initated by release of MMPs
basal cell at wound margin flattin and lose microvilli
growth factors stimulate production:
1) fibronectin -> laid down on denuded area (red carpet), acts as provisional matrix
2) integrins -> receptors for ECM proteins
wound healing - epithelium phase 2
Migration (24-36 hrs)
-triggered by cytokines and growth factors
- flattened basal cells move to cover wound with monolayer of cells -> bind to fibronectin matrix
- > pulls sheet of cells along fibronectin carpet step by step
- cell movement is energy dependent requiring protein and glycogen synthesis and Ca++
- single layer of cells cover wound -fill in
- no cell mitosis in or around the wound area until wound covered
wound healing - epithlium phase 3
proliferation (24-30 hrs)
limbal stem cells proliferation increasingly
basal cells differentiate into wing and squamous cells
- zonula occludens reform in superficial squamous layer
- epithelium regains normal thicknesss
BM remodeled by secretion of laminin that can attach to integrins
wound healing - epithelium phase 4
attachment
-reestablishes firm attachment of basal cells to BM and stroma
if BM not damaged, can occur in matter of days
limbal stem cell deficency
if destruction of stem cells -> conjunctivalization of cornea => vascularization and goblet cells
wound healing - epithelium summary
cell migration - sliding
cell proliferation - basal layer
cell differentation - normal structure restored
regulated by interaction b/n epithelial cells and extracellular matrix (BM)
- Type 4 collagen, laminin and fibronectin: regulate adhesion and migration
- growth factors and cytokines: stimulate growth, proliferation, migration, differentiation, adhesions, ECM despotision
damage to epithelium and basement membrane
regenerate basement membrane
form adhesion complexes: 4-6 weeks to months
can be susceptible to recurrent corneal erosion
basement membrane important to sequeseter cytokine and form stabel adhesion complex
wound healing - bowman’s layer
acellular
no regeneration
collagen scar (collage from stromal keratocytes filled in)
Wound healing - epithelial stroma interaction
epithelium interacts chemically with keratocytes via cytokines to mediate stromal healing
IL-1: master regulator of corneal wound healing
- released injured epithelial cells => keratocyte apoptosis
- modulate MMPs
- modulate growth factors
- prompt infammatory response
wound healing - stroma
destructive phase
removal of abnormal tissue
release chemokins that attract immune cells from limbal blood supply and tears
neutrophils as part of innate inflammatory response
-> release proteolytic enzymes and MMP to digest necrotic tissue
macrophage come later
platelet necessary for re-epithelialization
wound healing - stroma syntehtic phase
residual quiescent keratocytes adjacent to wound are activated
activated keratocytes repopulate wound site
-proliferation and migration
keratocyte migration is proposed to occur when epithelium has resurfaced the defect
subpopulation of activated keratocytes transformed by TGF-beta and PDGF into myofibroblasts
myofibroblasts:
- motile contractile cells
- deposit provisional ECM
- generate contractile forces to close wound
- remodel wound ECM (actin synthesis for motility, new collagen synthesis after debris cleared)
wound healing - stroma with excessive myofibroblast activity
contraction of corneal wound may cause irregular corneal astigmatism
repair tissue opacity
wound healing - stroma remodeling phase
ECM slowly restructured - weeks to years
regulated by TGf-beta and proteases
myofibroblast disappear
keratocytes become quiescent
wound healing stroma
summary
cellular interactions during corneal repair
upon corneal epithelial injury, IL-1 is released from injured epithelium into stroma
IL-1 induces some of the underlying stromal keratocytes to undergo cell death, while others induced to proliferate, secrete MMPs, and transition form quiescent to an activated phenotype
due to absence of basement membrane, corneal epithelial cells also secrete TGF-beta2 into underlying stroma inducing a subpopulation of keratocytes to undergo transformation into myofibroblasts that secrete ECM
the return of the basement membrane inhibits the release of TGF-beta2 into stroma and myofibroblast phenotyp no longer observed
the acitvated keratocyte continue to secrete IL1 and remodel the ECM
wound healing - corneal nerves
nerves regenerate from uninjured peripheral nerve trunks
rate of recovery = slow af
denervated cornea risk for epithelial defects
wound healing - descemet’s membrane
limited elastic properties
anterior banded portion - at birth
posterior portion - thorughout life
syntehsized collagen with less regular arrangement of collagen
appears as a scar/opacity
wound healing - endothelium
no mitosis in adult
defect covered by enlargement and spreading of neighboring cells - polymegathism, pleomorphism
Cornea wound healing summary
Epithelial cells: respond immediately to reestablish barrier fxn and protect underlying tissue from microbial organisms and dehydration
bowman’s membrane: no mechanism
stroma: corneal keratocytes (fibroblast) produce new reparative collagen and proteoglycans. repair tissue not transparent
descemet’s membrane: eventually resecreted by endothelial cells
endothelial cells: migrated and spread to cover defects - no mitosis
Photodamage
can occur with UV-a, UV-b, UV-c
mostly filtered by cornea
UV, violet and blue light trigger can trigger ROS by oxidative stress
antioxidant defense mechanisms
nonenzymatic antioxidants: ascorbic acid (primary antioxidant in cornea), glutathione, alpha-tocopherol
enzymatic antioxydant: corneal crystallins (ALDH3A1), catalase, glutathione peroxidase, superoxide dismutase, aldehyde dehydrogenase superfam
photodamage clinical effects
overexposure to UV light => epithelial injury
- inhibition of mitosis, loosening of epithelial layer
- nuclear and cytoplasmic damage - apoptosis with sloughing
- SPK - superficial punctact keratitis (dry eye)
acute photodamage
snow blindness, welding, tanning sun lamps
-symptoms: discomfort followed by pain and photophobia
chronic photodamage
pinguecula and pterygium
characteristics of corneal degenerations
deterioation and decrease in function
unliateral or bilateral -> asymmetric
no inheritance pattern
many degeneration considered aging changes
progression variable
often begin peripheral
general aging changes
1) decrease corneal thickness
2) increase descemet’s membrane thickness
3) decrease endothelial cell count
4) decrease corneal luster
arcus senilis
high cholesterol deposit at periphery of cornea
shrp peripheral border ending at edge of bowman’s layer with clear zone to the limbus
pterygium
proposed reactive fibrovascular response of conj strom to chronic irritants (especially UV)
invades superficial cornea, pinguecula does not
limbal girdle (of vogt)
symmetric yellow to white band in intrapalpebral limbals
2 presentations
1) white band with holes and narrow clear zone approaching limbus
2) chalky band with no holes or clear interval to limbals
increase with age
seen at 3 oclock and 9 oclock
guttata (not tested)
“droplet like” beaten-metal appearnce of descemet’s membrane with abnormal basement mebrane
brownish pigmentation
associated with fuch’s endothelial dystrophy
hassal-henle bodies (not tested)
focal thickenings of descemet’s membrane in peripheral cornea
common agin changes
crocodile shagreen (not tested)
asymptomatic degeneration in elderly
mosaic pattern
usually bilateral
