An Introduction to Corneal Physiology

Sara M. Thomasy DVM, PhD, DACVO

With appreciation for slides provided by Ellison Bentley and Christopher J. Murphy

Corneal physiology

• Multiple layers

• Major functions:

– Protection

• Biodefense

• Healing

– Refractive system

• Transparent

Corneal Anatomy

• Epithelium: stratified squamous non-keratinized

• Anterior basement membrane: Basement membrane of epithelium

• Bowman’s layer: collagenous acellular zone

– Most variable element across vertebrates (present in birds, most primates, lizards, elephant)

– Not present in domestic mammals

• Stroma: Lamellae composed of fine, uniform diameter, evenly spaced, collagen fibrils

• Descemet’s membrane: Robust basement membrane of endothelium

• Endothelium: simple cuboidal epithelium

anatomy.iupui.edu/courses/histo_D502/ Lab.19.eye.f01.html

Human/Primate/Bird : 6 layers

AE E

AE = Anterior epithelium

ABM = Anterior basement membrane

BL = Bowman’s layer or Anterior Limiting Lamina (acellular)

S = Stroma (with intersperced keratocytes)

DM = Descemet’s membrane

E = Endothelium

ABM BL S DM

www.vetmed.ucdavis.edu/.../ epath_overview_index.html

Domestic Mammals: 5 layers

No Bowman’s Layer

Dog/Cat/Horse/Cow: 5 layers

ABM

• Stratified, squamous non-keratinized epithelium

• Physical, molecular barrier to injury

• Smooth optical surface

• Self renewing over 7-14 day period

Adler’s 11th ed, 4.21

Anterior Epithelium

Dog: H&E

Anterior Epithelium

• ~10% of corneal thickness.

• 2-4 layers of superficial

squamous cells

• 2-8 layers of polygonal wing cells

• Single layer of basal cells

• Transparent due to few organelles

Horse:

Masson’s

trichrome

Squamous

Wing

Basal

Types of cell-cell connections

• Gap junctions (macula communicans)

– Arrays of small channels

– Permit small molecules to pass through

• Tight junction (zonula occludens)

– Fusion of membranes

– Near impermeable barrier

• Desmosome/hemidesmosome

– Convergence of tonofilaments at CM

– Dense fibrous material between

– Tissues that undergo mechanical stress

From Krachmer, Cornea fig 1-4

Junctional Complex

• Tight junction (zonula occludens)

• Zonula adherens

• Desmosome

If you got real small and looked at your

feet while standing here………

The most superficial cells of the anterior corneal

epithelium possess microplicae and microvilli.

Name one function these could play?

Adherence and stability of the PTF

Exchange of oxygen & nutrients between PTF and cells

Continual Loss of Superficial Squamous Cells

Left: SEM of multiple superficial squamous cells of the anterior corneal epithelium

Right: TEM of a single squamous cell of the anterior corneal epithelium sloughing

off the surface into the precorneal tear film. Where will it go?

http://80.36.73.149/almacen/medicina/oftalmologia/enciclopedias/duane/graphics/figures/v7/0080/004f.jpg

Superficial squamous cells

• Flat, polygonal

– 2-6 mm thick, 40-60 mm diameter

• Terminally differentiated

• Microvilli - increase surface area

• Less mitosis and metabolism

Note lack of nuclei in superficial cells (Adler’s 10th ed, fig 4-3, TEM)

Superficial squamous cells

• Smaller light cells – younger

• Larger dark cells - older, soon to slough

• Membrane associated mucins -

glycocalyx (from epi and goblet cells)

Transmission electron microscopy

Krachmer, Cornea vol I, fig 1-2, 1997

Dark cell

Superfical squamous cells

• Barrier function:

– Desmosomes & tight junctional complexes between cells

– Cell membranes joined together to form semi-permeable barrier

Surface epithelial cells of human cornea

From: Adlers’ 10th edition (fig 4-2,

D=desmosome, M=microvilli)

How would this

affect topical ocular

drug delivery?

Drug penetration through epithelium

• Intermediate solubility ideal

• Lipophilic drugs (CSA)

– Poor stromal penetration

• Hydrophilic drugs (Gentamicin)

– Poor epithelial penetration

Much more from Dr.

Clode in pharmacology!

Wing cells

• 2-4 layers between superficial & basal cells

• Distinct keratin expression

• Interdigitated

– Desmosomes, gap junctions connect

Basal epithelial cells

• Single cuboidal layer

• Gap junctions, junctional complexes

– Communication between cells

• Lateral interdigitations

• Mitotic activity Normal canine cornea

Basal epithelial cell attachment

• Hemidesmosomes-composed of integrins

– Receptors for ECM molecules

– Role in wound healing?

• Linked to keratin filaments and anchoring fibrils

– Type VII collagen

TEM of canine cornea

Hemidesmosomes

Anchoring

fibrils

Basal epithelial cell attachment

• Adhesion complex:

– Hemidesmosome

– Basement membrane

– Anchoring fibrils

– Anchoring plaques

• Basal cells rupture rather than

break adhesion complex

Spontaneous chronic corneal epithelial

defects (SCCED)

• Anterior stroma

– Thin hyalinized layer (4-5mm)

– Barrier to reformation of

adhesion complexes

• Rest of cornea normal on

light & electron microscopy

– NOT a basement membrane

abnormality

Is this a basement membrane defect??

Alterations in SCCED

TEM spontaneous chronic

corneal epithelial defect (SCCED),

magnification 29K

TEM of normal dog after 8

weekly debridements,

magnification 29K

Basal epithelial cells

• Cells move from outside in

• Stem cells

– Transient amplifying cells

• Replicate rapidly

• Limited number of times

• Become basal epithelial cells

– Move in and up

– Differentiate in process

Sloughed Cells are Replaced by Regeneration

From Limbus

The stem cells of the anterior corneal epithelium are located at the limbus

Limbal stem cells

• Corneal stem cells

– Differentiate into transient amplifying cells & one stem cell

– Reside palisades of Vogt

– No keratin K3

– Unique markers (p63)

– Unlimited life span

Limbal stem cell deficiency

Limbal allograft

Limbal allograft – 3 weeks post-op

Limbal allograft – 6 months post-op

What does this look like?

Limbal autograft – 2 months post-op

Symblepharon: A limbal stem

cell deficiency?

Physiologic roles of epithelium

• Optically smooth surface

• Hyperplasia of epithelium

after ablation of stoma

• Epithelial thickness may

be controlled by stroma

Immed post 4 months post

Physiologic roles of epithelium

• Barrier formation

– Tight junctional complexes of superficial cells most critical

– Drug implications:

• Must penetrate hydrophobic epithelium & hydrophilic stroma

Wild Type Klf4CN

Physiologic roles of epithelium

• Barrier formation

– Tight junctional complexes of superficial cells most critical

– Drug implications:

• Must penetrate hydrophobic epithelium & hydrophilic stroma

Wild Type Klf4CN

Physiologic roles of epithelium

• Barrier formation

– Tight junctional complexes of superficial cells most critical

– Drug implications:

• Must penetrate hydrophobic epithelium & hydrophilic stroma

Physiologic roles of epithelium

• Immunologic:

– Langerhans cells

• Specialized dendritic cells derived from bone marrow

• Reside among basal cells of peripheral epithelium

• Professional antigen presenting cells

• ↑ with ocular inflammation & migrate towards site of injury IVCM of equine cornea with

fungal keratitis

Langerhans cell in mouse

cornea

Primate

What structure is this?

Anterior basement membrane

What histologic stain is being used?

Periodic Acid-Schiff (PAS)

Anterior basement membrane

• Specialization of extracellular matrix

• 40-60 nm thick

• Type IV collagen (lamina densa)

• Laminin, fibronectin

• Epithelial and stromal sources

• Ligands for integrins

• Growth factors, proteoglycans

• Topography influences cell behavior

• Maintenance of epithelium

• Matrix for cell migration

SEM of canine BM

Anterior basement membrane

Abrams et al, Cells, Tissue, Organs 2002

An Intact Basement Membrane

Promotes Corneal Wound Healing

• Normal basement membrane:

– Usually difficult to remove

– Matrix for cell migration

– New adhesion complexes (days to

weeks)

• Removal of basement membrane:

– Longer to reform adhesion complexes

– May not be normal for >1 year

TEM Canine ABM

Normal canine basement membrane

Exposed stroma in SCCED patient

Primate

What structure is this?

Bowman’s Layer

Name 3 animals that have this layer.

Primates, birds, giraffes, & whales

What is the purpose of Bowman’s layer?Invest Ophthalmol Vis Sci. 2011 Nov 11;52(12):8818-27.

Invest Ophthalmol Vis Sci. 2011 Nov 11;52(12):8818-27.

• Transparent, refractive surface

• 80-90% of cornea

• Arranged in lamellae

• Major cell type: Keratocyte

• Mainly composed of ECM:

– Collagens I, III, V, VI

– Proteoglycans

– Elastin

Corneal Stroma – Substantia Propria

Canine corneal lamellae

(polarizing photomicrograph)

Stroma composed of

numerous lamellae

Endothelium on

Descemet’s membrane

• Collagen fibers are 25 nm

• Type I collagen primarily

• Lamellae – type I collagen bundles

• 300 in center

• 500 in periphery

• 2 mm thick & 960 mm wide

• Type IV collagen holds them together

• Parallel within lamellae but

orientation varies

• Circumferential annulus

Human corneal lamellae

Basement membrane 7.5Bowman‘s layer 110 Anterior Stroma 33

Descemet‘s membrane 50

Epithelium Not assessed

Endothelium Not assessed

Human

(kPa)

Posterior Stroma Not assessed

Rabbit

(kPa)

0.574.5Absent1.1

0.3811.74.1

Human vs Rabbit Cornea

Highly

Intertwined

Mostly

Parallel

Human Rabbit

Corneal collagen fiber organization in rabbits and dogs is similar

Elastic modulusAnterior stroma 1.1 kPa

Posterior stroma 0.48 kPa

Dog

Lamellar Structure of the Normal Dog CorneaJamie Jester; UC, Irvine: 2nd harmonic imaging

Stroma – proteoglycan structure

• GAGs bound to core proteins

• Named for protein cores

– Four types of proteins

• Lumican

• Keratocan

• Mimecan

• Decorin

– Two types of GAGs

• Keratin sulfate

• Dermatan sulfate

• Highly sulfated, negatively charged

Stroma – proteoglycan function

• Decorin-dermatan sulfate

– Binds to type VI collagen

– Anterior > posterior cornea

• Retains more water

• Lumican-keratan sulfate

– Important for transparency

• Keratocan-keratan sulfate

– Rarely found elsewhere than the cornea

• Mimecan-keratan sulfate

– Similar to a proteoglycan found in bone

• Responsible for spacing, packing of collagen fibrils

• Lumican plays role in collagen fibril diameter

• Binds Na+ & K+, decreases effective osmolarity

• Keratocan – important for corneal thickness

• Increase viscosity

Stroma – proteoglycan function

WT Kera KO

Keratocytes

• Similar to fibroblasts

• Stellate shape

• Structural & biochemical homeostasis

• Extensive cytoskeletal components

– Contraction

– Maintenance of corneal shape

• Dendritic processes-up to 50 mm long

Nishida T, Yasumoto K, Otori T, Desaki J. The network structure of corneal fibroblasts in the rat as revealed by

scanning electron microscopy. Invest Ophthalmol Vis Sci. 1988 Dec;29(12):1887-90.

Keratocytes Form an Interconnected Network that extends Throughout the

Entire Thickness of the Cornea

• Gap junctions between

• Function as a single cellular system

• Regulated by extracellular environment

– Interact with ECM through intergrins

• Turnover every 2-3 years

• Injury - become independent from each other

Keratocytes

• Synthesize:

– Collagens

– Glycosaminoglycans

– Matrix metalloproteinases

– Phagocytose foreign

material when activated

• Critical for stromal

wound healing

Keratocyte function

From Krachmer, 1997,Cornea fig 1-8

Other stromal cells

• MHC + antigen presenting cells

– Anterior stroma

• MHC - antigen presenting cells

– Peripheral and central stroma

• Macrophages

– Posterior stroma

• Adult stromal stem cells

– Periphery

Adler’s 11th ed Fig 4.13

Is the most posterior stroma unique?

• Thin strip of stroma after BB technique

• 5-8 lamellae

• Acellular, ‘strong’

• Proposed new layer

Ophthalmology 2013;120:1778-1785.

• But this area is not acellular

• Interlamellar adhesive strength known

• Why only visible on dissected samples?

Descemet’s

membrane

Ophthalmology 2013;120:1715-1717.

“There is nothing new

under the sun”

Corneal Transparency

• The hallmark of corneal disease is an opacity

• It’s transparent because of

– What it has

• Nonkeratinized epithelium

• Relative stromal dehydration

• Small uniform diameter collagen fibrils

– Evenly spaced

– Arranged in lamellae

– And what it doesn’t have

• Blood vessels

• Myelinated nerve fibers

• Pigmentwww.snopes.com/horrors/ techno/cornea.htm

That’s what we tell the students…

• But:

– Sizes of fibers do vary

– Spaces between fibers vary

– Bowman’s layer

• Transparent

• Fibers randomly arranged

Porcine cornea

Stromal transparency

• Control distance between fibrils:

– Proteoglycans

– FACIT collagens

– Type VI collagen

– Fibronectin

• Fibers connected to next nearest neighbor

– Resistant to AP & lateral stretching

– Lattice maintained

Müller L, 2004, EER

Stromal transparency

• Importance of fiber diameter:

– Artificial corneas

• Synthetic polymers and collagen

• Transparency dependent on diameter below wavelength of light

– Sclera

• Larger diameter

• Opaque

Biosynthetic corneal implant

Cornea

Sclera

What about stromal cells?

• Dendritic processes of cells – minimal light scatter

• Keratocyte nuclei – light scatter

• High concentrations of corneal crystallins in keratocytes

– Minimize fluctuations in refractive index

Normal canine cornea

Corneal crystallins

• Crystallins: water-soluble proteins

• Corneal epithelium & keratocytes

• Protective function

– Absorb UV radiation

– Decrease lipid peroxidation & free radical formation

• Major types:

– Aldehyde dehydrogenase (ALDH)

• 10-40% of the water-soluble protein in the cornea

– Transketolase (TKT)

What happens with corneal wounding?

• Dramatic increase in light scatter by myofibroblasts

• Marked decrease in ALDH and TKT

• Cellular basis for transparency?

Jester JV, Moller-Pedersen T, Huang J et al. The cellular basis for

transparency: evidence for ‘corneal crystallins.’ J Cell Sci, 1999: 112,

613-622.

AKA Posterior limiting lamina or posterior limiting membrane (PLM)

Endothelium or posterior epithelium

Descemet’s membrane or PLM

Posterior stroma

Descemet’s membrane

• True basement membrane of endothelium

• 7-15 mm thick

• Grows throughout life of individual

• Ends in anterior trabeculae of outflow angle

• Comprised of:

• Collagen types III, IV, V, VI & VIII

• Laminin

• Distinctively layered

• Resistant to proteolytic enzymes

• Tears with shearing forces

• Adherent to posterior cornea

Descemet’s membrane

What happens if Descemet’s membrane ruptures?

Corneal hydrops!

Feline acute bullous keratopathy - any risk factors?

Systemic cyclosporine administration Pierce et al Vet Ophthalmol 2016

Corneal endothelium: a simple cuboidal epithelium.

The innermost layer of the cornea. In contact with the

aqueous humor of the anterior chamber.

Bonus round: What other structures in nature are

packed in hexagonal arrays and why?

Corneal endothelium: a simple cuboidal epithelium.

The innermost layer of the cornea. In contact with the

aqueous humor of the anterior chamber.

Bonus round: What other structures in nature are

packed in hexagonal arrays and why?

Hexagonal geometry is the most efficient packing geometry in nature

Corneal Endothelium serves both barrier and active pump functions to

minimize water content of the stroma.

SEM of corneal endothelium that lines the

innermost surface of the cornea

• High metabolic activity

– Large nuclei

– Rich in mitochondria & Golgi apparatus

– Prominent endoplasmic reticulum

• Tight and gap junctions present

Does epithelium or endothelium contribute more to stromal dehydration?

From Birth to Death: It’s a constant loss of cells

Very poor regenerative capacity in most species (rabbit an exception)

That’s what we tell the students…

• How does endothelial cell density (ECD) decline with age?

• Cats

- Rapid, non-linear ↓ in ECD for first 10 months postnatal

- Slower decline from 10-12 months

- No change in ECD from 1-14 years

Cat

Barody RA, Bito LZ, DeRousseau, Kaufman PL. Ocular development and aging. 1.

Corneal endothelial changes in cats and in free-ranging and caged rhesus monkeys. Exp

Eye Res 1987 45:607-622.

That’s what we tell the students…

• How does endothelial cell density (ECD) decline with age?

• Cats

- Rapid, non-linear ↓ in ECD for first 10 months postnatal

- Slower decline from 10-12 months

- No change in ECD from 1-14 years

• Rhesus macaque

– 20% ↓ between 1-6 years

– 0.1-0.7% ↓ per year for life

– No difference caged versus free range

• Divide in rabbits

• Evidence for peripheral stem cells

Rhesus macaque

Barody RA, Bito LZ, DeRousseau, Kaufman PL. Ocular development and aging. 1.

Corneal endothelial changes in cats and in free-ranging and caged rhesus monkeys. Exp

Eye Res 1987 45:607-622.

How does increased IOP from glaucoma affect

the cornea?

• ↑ pressure gradient will force more fluid across an intact barrier

• ↓ corneal endothelial pump efficiency

• Will not directly disrupt the epithelial or endothelial barrier functions

Horse

What happens to the endothelium with injury?

• Polymegathism - variability in cell area

– Increased cellular stress, instability, abnormal cytoskeleton

• Pleomorphism - deviation from normal shape

• Both increase with age, corneal thickness does not

– Changes indicate stress, may be more likely to be damaged

Normal 47 yr old 47 yr old after 27 years of contact lens wear

Endothelial control of corneal thickness

• Ion transport systems

– Water moves osmotically down gradients from active ion transport

• Results in corneal dehydration

• Na+-K+ ATPase

• Na+-H+ exchanger

– Located in basolateral aspect of cells

– H+ acidifies aqueous & intracellular CO2

• Net result of 2 pumps

– Na+ out, K+ in, H+ out

• Carbonic anhydrase

– Coverts to H2 & bicarbonate→ moved out of cell→ water follows

• Mimic aqueous components

• Energy source = glucose

• BAB/junctional stabilizer = calcium & glutathione

• Buffer = bicarbonate (ion transport)

• pH = 7.38

• Osmolarity = 304

Intraocular irrigating solutions

Corneal Innervation It does more than just transmit nociception

• The most densely innervated structure in the body

• Nerves present anterior but not posterior

• Ophthalmic branch of CN V

– Sensory, autonomic nerve fibers

• Neuropeptides

– SP, CGRP, VIP

• Neurotransmitters

– Norepi & Ach

These compounds, secreted by the nerve endings, are trophic

(neurotrophic). They are essential for maintaining normal corneal health.

If you denervate the cornea, degenerative changes will occur!

Normal dog

360 mm 120 mm

Marfurt, IOVS 2001

Canine corneal innervation

• 14-18 superficial stromal nerve bundles enter radially at limbus

• Bundles branch

– dense anterior stromal plexus

• Epithelial leashes extend through basal layer, then through basal, wing, squamous cell layers

Marfurt, IOVS, 2001

Corneal wound healing

• Epithelial, stromal, & endothelial processes differ

• Trauma, infection, surgery

• Regulated by growth factors, cytokines, proteases, neuropeptides

– Autocrine, paracrine

– Epithelial cells, keratocytes, nerves, lacrimal gland, immune system

Corneal epithelial wound healing

• Cell migration

– Ameboid movement toward center of wound

– Cells thin

– Migrate toward center of lesion

• Centripetal movement

• 20-50 mm/hr

Adler’s Physiology, 10th edition, fig 4-26

• Initial lag phase

– Retract, thicken, lose hemidesmosomal attachments

– Cytoskeletal proteins, vinculin, actin, talin, integrins synthesized

– Integrins disassociate & migrate over surface

• Adherens molecule to ECM

• Fibronectin = provisional matrix for attachment

• Cell proliferation to re-establish thickness, differentiation,

adhesion complexes to BM

• Restore barrier & optical function

Krachmer, Cornea, Fig 8-7

Corneal epithelial wound healing

What’s wrong with SCCEDs?

• Have fibronectin

– Provisional matrix for cell attachment

– Epithelial cells can migrate

• No laminin, collagen IV, collagen VII

– Epithelial cells cannot reform rest of BM & adhesion complexes

Stromal wound healing

• Trauma, infection, inflammation

• Epithelial or stromal injury

– Adjacent keratocytes die

• Defect fills with fibrin clot

• Remaining keratocytes transform

Krachmer, Cornea, Fig 8-15

• Keratocyte-fibroblast-myofibroblast transformation

– Lose connections to other keratocytes

– Stimulated by TGFb

– Critical for wound contraction & matrix organization

– Myofibroblast phenotype

• Prominent actin filaments

• Expression of smooth muscle form of actin

Stromal wound healing

Stromal wound healing

• Polymorphonuclear leukocytes arrive in 2-6 hours

– Debride wound

– From tear film immediately & vasculature

• Myofibroblasts - migrate to wound margin

– Secrete extracellular matrix constituents

• PMNs & myofibroblasts ∆ fibrin plug to scar

Stromal wound healing

• Wound strength increases for 4-6 months

– Some studies-up to 4 years

• Scar composition

– Type III collagen

– Poorly sulfated proteoglycans

– Fibronectin

– Laminin

Krachmer, Cornea fig 8-20

• Corneal scarring:

– Different amounts & types of collagen, proteoglycans

– Differing diameters collagen fibers

– Increased reflectivity of keratocytes

Stromal wound healing

Keratomalacia

• Enyzmatic degradation

– Infection

– Inflammation

• Source:

– Bacterial or fungal collagenases

– MMPs from keratocytes or

inflammatory cells (PMNs)

• Matrix metalloproteinases (MMPs)

– Zinc binding proteases

– Collagenases (MMP-1, -8, -13)

– Gelatinases (MMP-2,-9)

– Stromelysins (MMP-3, -10, -11)

– Matrilysin (MMP-7)

• Degrade or modify ECM

• Tissue inhibitors of MMP-TIMPs-play a role in wound healing

• Need balance for normal wound healing to occur

Keratomalacia

Endothelial wound healing

• Defects primarily covered by cell spread

– Leads to cell enlargement

– Do not break contact

– Some myoblastic differentiation

– Maybe some endothelial proliferation at wound edge (cats)

– Corneal thickness not normal until monolayer re-established

Epithelial-stromal interactions

• Complex environment

– Soluble factors

– Extracellular matrix

– Biomechanical factors

• Studying factors in isolation

– Likely inaccurate

Epithelial-stromal interactions

• Epithelial debridement/injury

– Apoptosis of underlying keratocytes

– Found in multiple species

– Inducible factor from tears?

Canine cornea 3 days post epithelial debridement

Growth factors in epithelial wound healing

• Growth factors - stimulate cell mitosis in tissues

– Named by tissue of origin or behavior

• Platelet-derived growth factor or transforming growth factor

• Cytokines - mitosis & differentiation of inflammatory cells

• Distinction is artificial

Questions?