Wound healing of cornea

WOUND HEALING OF
CORNEA
Panit Cherdchu
Department of ophthalmology
Phramongkutklao Hospital

OUTLINE
NORMAL HISTOLOGY
CORNEAL WOUND HEALING
STROMAL WOUND HEALING
ENDOTHELIUM WOUND HEALING
CONCLUSION
OUTLINE

“The cornea is a transparent, avascular tissue
that measures 11-12 mm horizontally and 10-11
mm vertically”

• Average radius of curvature :
7.8mm
• Major source of astigmatism
• Depends on glucose from
aqueous humor and oxygen
diffusing through tear film
• Peripheral area is supplied by
limbal circulation

• Highest densities of nerve
endings
• Sensory nerve fibers from
long ciliary nerves and
form a subepithelial plexus
• Neurotransmitters in the
cornea : Ach,
cathecolamines, substance
P, calcitonin gene-related
peptide, neuropeptide Y,
interstinal peptide, galanin
and methionine-
enkephalin

• Central cornea thickness 540 micron (thicker
towards the periphery)
CORNEACROSSECTION

EPITHELIUM
• The epithelium+tear film forming smooth
surface
• Stratified squamous cell
• Tight junctions
• Microvilli on their outermost
surfacedesquamate into tears (7-14days)
• Tight packinguniform refractive index+light
scattering⬇⬇

BOWMAN LAYER
• smooth, acellular, nonregenerating layer, located
between the superficial epithelium and the stroma
• It is composed of strong, randomly oriented collagen
fibrils
• In adult humans, Bowman's membrane is 8-12
microns thick. With aging, this layer becomes thinner

The Bowman's membrane is
named after Sir William
Bowman (1816–1892),
an English physician,
anatomist and
ophthalmologist, who
discovered this membrane.

STROMA• Keratocytes vary in
density and size
• 3-dimensional
network
• Flatten fibroblasts
• Keratocytes declines
with age, surgery

• Stroma is composed of an extracellular matrix
formed of collagens and proteoglycans.
• Type I, V fibrillar collagens + type VI collagen
• Decorin and lumican
• Posterior stroma is wetter than the anterior
• Human cornea has little elasticity and
stretches only 0.25% at normal IOP

DESCEMET MEMBRANE
• Lies betwenn stroma and endothelium
• Its thickness ranges from 3 μm at birth to 8-10
μm in adults.
• The corneal endothelium is a single layer of
squamous cells covering the surface of the
cornea that faces the anterior chamber

• It was named after
French physician
Jean Descemet
(1732–1810).

• Descemet's membrane is
also a site of copper
deposition in patients with
Wilson’s disease or other
liver diseases, leading to
formation of Kayser-
Fleisher rings

ENDOTHELIUM
• Interdigitated cells arranged in a mosaic
pattern of mostly hexagonal shapes
• Cell loss results in enlargement and spread of
neighboring cells
• Normal density is 2000-3000 cell/mm2

• Cornea Clarity is maintained by 2 functions
– Active as a barrier to the aqueous humor
– Providing a metabolic pump

TEAR
• Protective layer to
wash away irritants
and pathogens
• Maintaining a
smooth optical
surface between
blinks
• Uniform gel (lipid,
aqueous and mucin
layers)

Latent phase
Cell migration
and adhesion
Cell
proliferation
4-6 hours
24-36 hours
Lasts 36 hours to
several months

Latent Phase
• Firsthemidesmosomes are lost, provisional
attachment complex is formed called focal
contactsepithelium cells flatten and migrate
as an intact sheet to cover the wound

• Secondcell distal to the original wound
proliferate to repopulate the wound area and
cell stratification and differentiation occur
Cell migration and adhesion

• Thirdhemidesmomsomes are reformed and
extracellular matrix synthesis and reassembly
occur
Cell proliferation

interaction• Complex cascade of events involving cytokine-
mediated interactions between the epithelial
cells, keratocytes stroma, corneal nerves,
lacrimal glands and cells of the immune
system

• Epithelial-mesenchymal interactions play a
vital role in wound healing

• Movement of existing
basal epithelial cells at
the corneal wound
margin
• 4-6 hours after corneal
injury
• PMN,Leukocytes from
tearremove necrotic
cells
Latent Phase

• Hemidesmosomal attachments between the
basement membrane and the basal cells completely
disappear to approximately 70 micron outward
from the ED margin
Latent Phase

• Cellular processes on the basal edges of cells
bordering the wound
• finger-like filopodia or wider shaped lamellipodia
Latent Phase

Epithelial injury
Reduction
inhemidesmosome
attachments
200micron outward
lamellipodial/
filopodial
extension
PMN,Leukocyte
invasion Removal of
necrotic
cells/debris
Retraction/rounding
of epithelial cells at
wound margin

• Intracellular formation of actin filaments
(fodrin,vinculin and ankyrin)
• Formation of filopodial and lamellipodial
extension completed
• Actinfilaments accumulate at leading edges of
lamellipodia and filopodia giving cytoskeletal
support
• Appearance of fibronectin(1hr)

• Temporary anchor formation cyclical process
commences as cells start to advance
• Centripetal migration of leading epithelial cells
across stromal surface. Energy derived from
glycogen metabolism

• Formation of Y-X contact lines
• Completion of epithelial monolayer covering wound area

• Fibronectin disappears
• Synthesis of new hemidesmosomes and other
anchoring complexes
• Appearance of type III collagen
• Epithelial/stromal adhesion restored from 6-8
weeks although abnormalities can persist for
up to 1 months

• Stem cells are located near limbus
• Wound near limbus is healed more quickly
• Palisade of Vogt ( the transitional zone)
Cell proliferation

• Activation of limbal stem cells
• Stem cells produce transient
amplifying cells (TACs)
• TACs give rise to post mitotic cells
(PMCs)
• PMCs give rise to terminally
differentiated cells (TDCs)
Cell proliferation
Stem cell
TACs
PMCs
TDCs

• Further establishment of hemidesmosomes
• Possible epithelial hyperplasia
• Replacement of corneal nerve axon terminal
endings
• Hypersensitivity of corneal epithelial nerves
for several months
Cell proliferation

Stromal wound healing
• Avascularity makes the slower wound healing
process
• Interaction between epithelial cells and keratocytes
where polypeptide growth factors play an important
role
• Keratocyte undergo proliferation and migration
stimulated by the release of certain cytokines

• Keratocyte activity starts after the wound is
completely covered with new epithelium
• Keratocytes fibroblastic transformation with
resulting expansion of the fibroblast population by
mitosis after 48-72 hours
• Peak at 3-6 days

• Fibroblasts produce collagens, glycoproteins and
proteoglycans which from the new stromal
extracellular matrix
• The human corneal stroma contains collagen type
I,III,V and VI

• Early phase, newly synthesized collagen fibrils are
usually larger in diameter than normal, due to higher
concentration of chondroitin/dermatan sulfate
which lasts for up to 3-6 months

• Stromal remodelling is controlled by various matrix
metalloproteinases (collagenase, stromelysin and
gelatinase)
• Removal of damaged collagen fibres is controlled by
the presence of PMN,Leukocytes and proteolytic
enzymes

Factor effect stroma healing
1.Several substances associated with the corneal
wound healing process activate keratocytes.
 Epithelial growth factor
 Fibroblast growth factor (FGF)
 Interleukin-1
 Transforming growth factor-β
 Insulin
 Retinoic acid

2. Epithelial-stromal interactions appear augment the
wound healing process by causing the production of
a thicker and stronger extracellular scar matrix than
that found in deeper stromal wound regions that
receive no epithelial cell factors

2. Epithelial-stromal interactions
 Hypercellular fibrotic stromal scar type in wound
regions where epithelial-stromal interactions occur
(strong, but can look clinically hazy )
 Hypocellular primitive stromal scar type in wound
regions where keratocyte injury pathways work alone

Astigmatic keratotomy Photorefractive keratotomy

LASIK 4 years after surgery

3.Realigned wounds
 sutured wounds or unsutured wounds with minimal
gaping and no epithelial cell plugging (well-aligned
wounds)
 wounds with wide wound gaping, epithelial plugging,
or incarceration of adjacent corneal tissue (poorly-
aligned wounds)

Corneal stromal wounds heal in two distinct phases
1. An active phase that results in the production of a
stromal scar (over the first 6 months after injury in
humans)
2. A remodeling phase that improves corneal
transparency and increases wound strength (occurs
up to 3.5 years after injury in humans)

Common ophthalmic procedures
 Cataract extraction, penetrating keratoplasty , radial
keratotomy ,PRK, LASIK. Clear corneal cataract
wounds are corneal stromal incisions constructed at
oblique angles to corneal surface so that they self-
seal
 They usually heal with well-aligned external wound
margins and wound edges subepithelial zone of
hypercellular fibrotic stromal scarring and a
remaining deeper zone of hypocellular primitive
scarring

Common ophthalmic procedures
In marked contrast, limbal and scleral tunnel cataract
incisions heal because fibrovascular granulation
tissue from the episclera completely grows into the
wound by 15 days after surgery and finishes
remodeling by 2.5 years after surgery

endothelium
• Crucial in maintaining stromal transparency
• No capacity to replicate by mitosis
• Largely dependant on enlargement and
movement of surrounding cells to cover a
wound site

• Responsible for the deposition of a new
descemet’s membrane
• Contains fibroblast-like cells, collagen fibrils,
basement membrane proteins and junctional
complexes (retrocorneal fibrous
membrane=RCFM)
endothelium

Endothelium of the rabbit following a
transcorneal freeze heals in 3 stages
Stage 1 (0 to 3 days) is characterized by an initial
coverage of the wound by pleomorphic
spindle-shaped cells that form a functional but
incomplete barrier and have minimal pump
site density
endothelium

Stage 2 (4 to 7 days) the cells assume a flattened
configuration, have an irregular polygonal
shape, and establish normal pump-site density
and barrier function.
Stage 3 (8 to 30 days) is characterized by a
continuation of the remodeling of the
monolayer
endothelium

Summary of corneal wound healing
1.EPITHELIAL PHASE.
12–48 hours
Surface epithelium begins to slide and
replicate, resulting in the formation of an
epithelial plug that fills the cavity
Although maturation of basement membrane
adhesion complexes may require up to 6
weeks

2.STROMAL PHASE
Lasts for several weeks
Migration of activated keratocytes into the
wound ,these keratocytes transform into
myofibroblasts
Bridge the gap with secondary contraction and
thus help to re-approximate the wound
margins
Synthesize and secrete new collagen

3.CROSS-LINKING AND INITIAL STABILIZATION
PHASE
The collagen forms cross-links over a period of
several months, which stabilizes and
strengthens the wound and secures it

4. REMODELING AND STRENGTHENING PHASE
Lasts for many months after surgery
Residual collagen synthesis and breakdown, as
well as continued collagen cross-linking, occur
during this phase. These processes help to
further strengthen the wound

Reference
Ophthalmology 2nd edition (September 2003)
by Myron Yanoff (Editor), Jay S. Duker (Editor),
James J. Augsburger (Editor) By Mosby
Duane's Ophthalmology
Basic and clinical science course 2015-2016
 CHRIS STEELE BSc (Hons), MCOptom, DCLPHEAD OF
OPTOMETRY, SUNDERLAND EYE INFIRMARY

Wound healing of cornea

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