OP: Corneal Epithelium/Stroma Development + Structure

What are the layers of the cornea? Furthermore, what tissues form every structure of the cornea? Lastly, when does development of it begin?

1. Epithelium (superficial/wing/basal)

2. Bowman’s

3. Stroma

4. Descemet’s

5. Endothelium

All tissues derived from ectoderm, specifically surface and neural crest.

After formation of the lens

Write out the rough timeline (without dates) of structure formation in the eye. Make sure to distinguish between neonatally and after birth.

Lens → Cornea → Endothelium (neonatally)

Stromal development (after birth)

How many rounds of epithelial thickening happen? When does each occur (stage of development), what one of them produce, and how much does each increase epithelium thickness by roughly?

2 rounds

Eye lid opening (neonatally):

• Increases epithelium to 4 cells thick

• Produces basement membrane and anchoring complexes to primitive stroma = hemidesmosomes

After birth:

• Epithelium thickens to 4-6 cells after birth

What is the believed origin of Bowman’s layer?

It is believed to be a primitive stroma remnant.

What is responsible for enabling primary stroma development? What are the 3 ways it does this and how is it produced?

Hyaluronic acid/hyaluronan

• Synthesized in large by primitive corneal endothelium and keratoblasts

• High negative charge

• Draws water in and causes stromal swelling

• Provides a substrate for further cell migration into the stroma

What causes secondary stromal development, when does it begin, and how does it change the corneal stroma?

• D/t continued maturation of the cornea causing removal of hyaluronans and dehydration (d/t endothelial pump function)

• Begins in utero

• Central cornea thickness decreases by about 5%

What breaks down hyaluronan? What does this culprit also produce?

Keratocytes produce and secrete hyaluronidase which destroys hyaluronan

• They also change secretion to synthesize collagen and other GAGs for forming lamellae.

What are the three factors of endothelium maturation and what is the function of each? Furthermore, describe the pathway of one of them (hint: stroma)

Tight junction formation limits paracellular water transport (water regulation - transparency)

Production and activation of endothelial pump proteins (secondary stromal development - transparency)

Stroma decreases water content (water regulation - transparency)

• Water/ions moved across endothelial cells into AH

• Stroma thins

• Cornea becomes transparent

Write out the 4 steps of cornea stromal development. Describe the 3 key events of the 2nd step and the 7 key events of the 4th step.

1. Mesenchymal cells migrate from limbal region beneath the corneal epithelium after lens invagination to form primitive endothelium and keratoblasts

2. After primary migration

   • Some collagen deposition

   • Keratoblasts produce hyaluronan and the primary stroma

   • Primitive endothelium begins producing Descemet’s membrane

3. Second wave of mesenchymal cells migrate from limbus and become keratoblasts

4. Dehydration/Compaction/Organization

   • Occurs in posterior anterior direction

   • Production of hyaluronidase

   • Production of mature GAGs

   • Collagen deposition

   • Organization of lamallae

   • Endothelial maturation

   • Transparency

What are the corneal changes of early life and adult/late life? What accumulation can occur late in life and does this affect vision?

Birth - 2 years:

• Cornea grows affecting optic power

Adult/Late life:

• Decrease in keratocyte number

• Decrease in corneal nerve fibers

• Decrease in endothelial cell density

• Increase in epithelial basement membrane thickness

• Increase in stiffness/strength of stroma

• Increase in descemet’s

• Impaired wound healing

• Arcus senilis (cholesterol deposits outside of the visual axis)

What shape is the collagen of the cornea? How does the size of this shape compare going from central → peripheral cornea? How about stroma vs bowman’s? Lastly, how does the spacing between collagen change from central to peripheral?

• Lamellae

• Central → Peri = increase in width and thickness of lamellae

• Stroma > Bowman’s

• Increase in spacing from central to peripheral cornea

How are the lamellae in the stroma of the cornea arranged in the anterior 1/3 vs the posterior 2/3? Why? Lastly, state the relative mechanical strength of the following structures: bowman’s, descemet’s, posterior 2/3, anterior 1/3.

Anterior 1/3: Woven like a basket for mechanical strength

Posterior 2/3: Non-woven oriented 45 degrees apart per layer for transparency

What are the two examples given of corneal ectasia? What is the etiology/symptoms of each? Are they unilateral or bilateral?

Keratoconus:

• Bilateral

• Progressive thinning

• Blurred vision (irregular astigmatism)

• Etiology unknown

Post-LASIK ectasia:

• Mechanical failure from weakened structure

• Interlamellar slippage

• Uni/bilateral depending on how many eyes get lasik

How are the lamellae of humans oriented in the stroma? What about adjacent lamellae? Lastly, what is the peripheral collagen organization and pathway (as far as location)?

Stroma has lamellae oriented orthogonally while adjacent lamellae lack this orthogonal orientation.

Peripheral collagen fibrils throughout the width of stroma stretch across the cornea and turn 90 degrees to form a circumferential annulus (looks like Genshin Impact star)

What are the two fibril forming + non-fibril forming collagen types? Make sure two name the two subtypes of non-fibril forming.

Fibril forming:

• I

• V

Non-fibril forming:

• VI

• FACIT (fibril associated collagens with interrupted triple helices - X + XIV)

What two collagens comprise banded collagen and what is the function of each collagen type? Lastly, what prevents additional segments from binding to the collagen band?

• V = limits diameter of collagen fibrils by introducing small mismatches

• I = binds to V

• Weaker covalent bonds prevent binding of additional segments

How is Elhers-Danlos Syndrome passed, what 4 genes does it affect, and what are the 6 presentations?

Genes:

• Collagen I

• Collagen V

• Non-collagenous extracellular matrix proteins

• Intracellular process genes

Presentations:

• Skin hyperextensibility

• Delayed wound healing

• Joint hypermobility

• Corneal thinning

• Stromal folding

• Keratoconus

What are the two functions of VI collagen and what does it look like?

• Binds corneal lamellae together

• Bridges other types of collagen

• Nonbanded and beaded

What are the three functions of keratocytes? How much of the stroma do they comprise relative to collagen, how does their density change with age (give relative % change), and how are they localized/organized?

• Produce collagen fibrils and ECM

• Control of turnover in stromal ECM

• Source of ECM in the event of injury

• Second most to collagen

• Decreases by nearly 50% from infant to adult

• More localized anteriorly with a radial organization

List the 5 steps of collagen formation. What is a require reactant as well for the first step?

1. Prolyl + lysyl hydroxylase modify proline + lysine to hydroxyproline+lysine within the endoplasmic reticulum

2. O-linked glycosylation on hydroxylysine residues

3. Intrapeptide disulfide bonds form between propeptide sequences to align 3 chains → drives triple helix formation

4. Procollagen peptidase cleaves N/C terminal peptides from procollagen triple helix

5. Following exocytosis, lysyl oxidase catalyzes linkage between tropocollagen units

Ascorbic Acid (Vitamin C) is required for hydroxylation of proline/lysine by their respective enzymes

What are fibropositors? How are they oriented and why?

• Long cytoplasmic extensions of keratocytes that surround nascent fibrils, depositing into forming lamellae.

• Orthogonally which helps produce the organized fibril/lamellar structure

What are the two categories of collagen fibril maturation? What are the two subcategories of one of them? Lastly, what does maturation cause and what happens if it occurs excessively?

• Crosslinking maturation

  • Enzymatic (divalent cross linking)

  • Non-enzymatic (trivalent cross linking)

• Glycation

• Maturation causes an increase in corneal stiffness d/t stronger and tougher fibrils

• Excessive maturation can cause brittleness

What are the two types of crosslinking, what is each due to, and what does each link?

Divalent Crosslinking

• d/t lysyl oxidase activity (molecularly alters lysine with an aldehyde)

• occurs between 2 collagen tripeptides (tropocollagen)

Trivalent Crosslinking

• Independent of lysyl oxidase (non-enzymatic)

• Links 3 collagen tropocollagens (aspartic acid/glycine)

• d/t spontaneous racemization/isomerization

Is glycation non-enzymatic? What does it utilize and what 3 types can it occur between? Lastly, is it reversible?

• Non-enzymatic

• Utilizes a reducing sugar (glucose)

• Occurs between

  • Different tropocollagens

  • Within the same tropocollagen

  • Different fibrils

• Irreversible

What are the three general molecules that can form AGEs? What is it’s purpose for collagen, what structures does it affect, how does it alter the tissue, and lastly how can it accumulate pathologically?

• Collagen/Lipids/Proteins

• Collagen

  • accumulates naturally with age (especially long lived collagen molecules like in the stroma)

  • accumulates pathologically with hyperglycemia

  • Affects vasculature, lens, vitreous, outer retina, and trabecular meshwork

  • Alters mechanical properties

    • Increased stiffness of tissues

    • Increased brittleness of tissues creating disposition to injury

• AGE = advanced-glycation end products.

What does UVA-Riboflavin do? What is it used to treat?

• Crosslinks corneal collagen

• Reduces the amount of LOX-induced crosslinks in keratoconus and other corneal thinning disorders in order to boost mechanical strength.

What are the two components of stromal proteoglycans? What are the 4 subtypes of the first component?

• Protein core

  • Decorin

  • Lumican

  • Keratocan

  • Mimecan

• Glycan chain

What is the glycan chain comprised of in stromal proteoglycans? What are its 5 functions?

• GAGs

• Spacer molecules between collagen fibrils

• Control fibril diameter growth, assembly, and caliber

• Resist compressive forces

• Grant viscoelastic properties of cornea

• Assist transparency

How are fibrils attached to proteoglycans? Where do these proteoglycans extend into? Lastly, what 2 things do stromal proteoglycans regulate in terms of fibrils?

• Non-covalently attached

• Extend into interfibrellar space

• Regulate fibril size + diameter

What are the two types of glycan chains? What proteoglycans are each comprised of and what are the two sugars that make up each? Lastly, which is more abundant?

Keratin sulfate:

• Lumican, keratocan, mimecan

• Disaccaride made up of galactose and N-acetylglucosamine

Dermatan sulfate:

• Decorin only

• Disaccaride made up of iduornic acid and N-acetylgalactosamine

Keratin > Dermatan

How abundant is keratan sulfate? What are the three proteoglycans that comprise it? Furthermore, what does it do, where is it most abundant, what environments possess it in the highest concentrations, and how does its water binding capability compare to that of dermatan sulfate? Why is its water binding capability less/more?

• 60% of total GAGs

• Lumican, keratocan, mimecan

• Regulates consistency and spacing of fibril diameter

• More abundant in posterior stroma

• More common in low oxygen environments

• Promotes cell migration during development

• Water binding capability is less than dermatan sulfate

• Less because water must exit the posterior stroma across the endothelium

What is the relative abundance of dermatan sulfate, what does it do, what is the primary proteoglycan within it, where is it most abundant (environmentally too?), and lastly how does its affinity for water compare to keratan sulfate. Why is it more/less?

• 40% of total GAGs

• Facilitates fibril spacing

• Decorin

• More abundant in anterior cornea

• Binds water with greater affinity than keratan sulfate

  • For resistance to evaporation

• More common in high oxygen environments

Where would you expect to see a large concentration of dermatan or keratan sulfate? What are the three properties about each of these tissues that explains why these proteoglycans are in high concentration there? Lastly, why is it important for each of these tissues to have this particular GAG within them?

Anterior Stroma:

• Dermatan sulfate

• High O2

• High NADH

• Increased water affinity

• Needed for evaporation protection (proximity to epithelium)

Posterior Stroma:

• Keratan sulfate

• Low O2

• High NAD+

• Decreased water affinity

• Endothelium needs to constantly pump water out

What determines which GAG is generated in a tissue? How does the environment a tissue exists in affect this factor?

• NAD+:NADH ratios determine which GAG is generated

• Anaerobic environment (posterior stroma)

  • More NAD+ available

  • Glucose concentrated to UDP-galactose which is used for generating keratan sulfate

• Aerobic environments (anterior stroma)

  • More NADH available

  • Glucose is alternatively shuttled to a different pathway to facilitate dermatan sulfate formation