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 Human/Primate/Bird : 6 layers

AEABM BL S DM 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 anatomy.iupui.edu/courses/histo_D502/ Lab.19.eye.f01.html E = Endothelium Dog/Cat/Horse/Cow: 5 layers

www.vetmed.ucdavis.edu/.../ epath_overview_index.html Domestic Mammals: 5 layers No Bowman’s Layer Anterior Epithelium • 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 Dog: H&E Anterior Epithelium

• ~10% of corneal thickness. Squamous • 2-4 layers of superficial squamous cells • 2-8 layers of polygonal wing cells Wing • Single layer of basal cells • Transparent due to few organelles Basal

Horse: Masson’s trichrome 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 are characterized by possessing microplicae and microvilli. Name one function these could play?

Adherence and stability of the PTF Exchange of oxygen & nutrient between PTF and cells Continual Loss of Superficial Squamous Cells

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

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? 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 Dark cell

• 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 Superfical squamous cells

• Barrier function: – Desmosomes & tight junctional complexes between cells – Cell membranes joined together to form semi-permeable barrier

How would this affect topical ocular drug delivery?

Surface epithelial cells of human cornea From: Adlers’ 10th edition (fig 4-2, D=desmosome, M=microvilli) 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 Hemidesmosomes of integrins – Receptors for ECM molecules – Role in wounding healing? • Linked to keratin filaments and anchoring fibrils Anchoring – Type VII collagen fibrils

TEM of canine cornea 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) Is this a basement membrane defect??

• 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 Alterations in SCCED

TEM spontaneous chronic TEM of normal dog after 8 corneal epithelial defect (SCCED), weekly debridements, magnification 29K 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 Immed post 4 months post be controlled by stroma 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 Langerhans cell in mouse IVCM of equine cornea with cornea fungal keratitis What structure is this?

Primate 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 Anterior basement membrane SEM of canine BM

• Ligands for integrins • Growth factors, proteoglycans • Topography influences cell behavior • Maintenance of epithelium • Matrix for cell migration

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 What structure is this?

Primate Bowman’s Layer Name 3 animals that have this layer. Primates, birds, giraffe, & whales Invest Ophthalmol Vis Sci. 2011 Nov 11;52(12):8818-27. What is the purpose of Bowman’s layer? Invest Ophthalmol Vis Sci. 2011 Nov 11;52(12):8818-27. Corneal Stroma – Substantia Propria

• 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 Canine corneal lamellae (polarizing photomicrograph) • Collagen fibers are 25 nm • Type I collagen primarily • Lamellae – type I collagen bundles • 300 in center • 500 in periphery • 2mm thick & 960 mm wide • Type IV collagen holds them together Stroma composed of • Parallel within lamellae but numerous lamellae orientation varies • Circumferential annulus

Endothelium on Descemet’s membrane Human corneal lamellae Human Rabbit (kPa) (kPa) Epithelium Not assessed 0.57 Basement membrane 7.5 4.5 Bowman‘s layer 110 Absent Anterior Stroma 33 1.1

Posterior Stroma Not assessed 0.38 Descemet‘s membrane 50 11.7 Endothelium Not assessed 4.1 Human vs Rabbit Cornea

Human Rabbit Highly Intertwined

Mostly Parallel Lamellar Structure of the Normal Dog Cornea Jamie 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 Stroma – proteoglycan function

• 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 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 Keratocytes Form an Interconnected Network that extends Throughout the Entire Thickness of the Cornea

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

• 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 Keratocyte function

• Synthesize: – Collagens – Glycosaminoglycans – Matrix metalloproteinases – Phagocytose foreign material when activated • Critical for stromal wound healing

From Krachmer, 1997,Cornea fig 1-8 Other stromal cells

• MHC + antigen presenting cells – Anterior stroma • MHC - antigen presenting cells – Peripheral and central stroma Adler’s 11th ed Fig 4.13 • Macrophages – Posterior stroma • Adult stromal stem cells – Periphery Is the most posterior stroma unique?

Ophthalmology 2013;120:1778-1785. • Thin strip of stroma after BB technique • 5-8 lamellae • Acellular, ‘strong’ • Proposed new layer Ophthalmology 2013;120:1715-1717. • But this area is not acellular • Interlamellar adhesive strength known • Why only visible on dissected samples?

Descemet’s membrane

“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 • Pigment www.snopes.com/horrors/ techno/cornea.htm Lattice theory: • Light scattered by individual fibers of uniform diameter cancelled out by adjacent light scatter • Requires absolute equal diameter, absolute spacing

Normal K9 eye Corneal Edema: • With water inbibition, the stroma swells disrupting the even spacing of the collagen fibrils. • Light is scattered and reflected resulting in the detection of a corneal opacity by the clinician.

2 y old dog with diffuse corneal edema secondary to glaucoma 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 Cornea • Larger diameter • Opaque

Sclera

Biosynthetic corneal implant 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 • Projective function – Absorb UC 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. Descemet’s membrane

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 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?

Hexagonal geometry is the most efficient packing geometry in nature 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.

• High metabolic activity – Large nuclei – Rich in mitochondria & Golgi apparatus – Prominent endoplasmic reticulum SEM of corneal endothelium that lines the • Tight and gap junctions present innermost surface of the cornea Does epithelium or endothelium contribute more to stromal dehydration?

↑ 500%

↑ 200% 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…

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 Rhesus macaque – No difference caged versus free range • Divide in rabbits • Evidence for peripheral stem cells

• ↑ 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 Intraocular irrigating solutions

• Mimic aqueous components • Energy source = glucose • BAB/junctional stabilizer = calcium & glutathione • Buffer = bicarbonate (ion transport) • pH = 7.38 • Osmolarity = 304 Corneal Innervation It does more than just transmit nociception

• The most densely innervated structure in the body • Nerves present anterior but not posterior Normal dog • Ophthalmic branch of CN V – Sensory, autonomic nerve fibers • Neuropeptides – SP, CGRP, VIP • Neurotransmitters – Norepi & Ach

360 mm 120 mm Marfurt, IOVS 2001 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! 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 • 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 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 Corneal epithelial wound healing

• Cell proliferation to re-establish thickness, differentiation, adhesion complexes to BM • Restore barrier & optical function

Krachmer, Cornea, Fig 8-7 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 Stromal wound healing • 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

• 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 Stromal wound healing

• Corneal scarring: – Different amounts & types of collagen, proteoglycans – Differing diameters collagen fibers – Increased reflectivity of keratocytes Keratomalacia

• Enyzmatic degradation – Infection – Inflammation • Source: – Bacterial or fungal collagenases – MMPs from keratocytes or inflammatory cells (PMNs) Keratomalacia

• 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 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?