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An Introduction to Corneal Physiology

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An Introduction to Corneal Physiology 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 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
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