What is the strength of human cornea?

What is the strength of human cornea?

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What is the difference of pressure a human eyeball cornea (cornea, lens and other elements separating vitreous body from air) can handle? Or asking differently: what the outside pressure should be to "blow up" a healthy man's eyeball?

Normal pressure in the eye1,2 is between 10 and 21 mmHg (somewhere around 1.3-2.8 kPa or 0.01-0.03 atm) with pathology occurring at around 30 mmHg.3 Clearly the cornea can handle that, but how much more? There's a lot more evidence as far as I could see for how well surgeries hold up, with 150-300+ mmHg (20-40 kPa, 0.2-0.4 atm).4 One source5 from 1998 cites work supposedly showing that the stroma:

gives the cornea 100 times more strength than is necessary to withstand the maximum intraocular pressure under physiological conditions.

So maybe around 200 kPa, for just the stroma? Interestingly, a new eye layer was discovered earlier this year; this Dua's Layer is incredibly thin - 15 microns - but can withstand pressures of around 100-200 kPa (750-1500 mmHg, 1-2 atm).6,7 The large range there is due to some conflicting numbers in those links; the 200 kPa is the more widely reported one, but again, that's just for one layer.

Then, of course, there is this: a beautiful 2009 thesis by Jill Aliza Bisplinghoff from Virginia Tech which thoroughly examines the issue. As an answer to your question, it includes the following such gems:

The high rate pressurization of 20 human eyes resulted in a mean rupture pressure of 0.97 ± 0.29 MPa (7275.60 ± 2175.18 mm Hg)… A student T-test revealed that the difference in the rupture pressure between the equatorial mean of 0.93 ± 0.30 MPa (6975.57 ± 2250.19 mm Hg) and the meridional mean of 1.13 ± 0.21 MPa (8475.70 ± 1575.13 mm Hg) was not significant (p=0.16)


A pneumatic cannon was used to impact eyes with a variety of projectiles at multiple velocities… The projectiles selected for the test series included a 6.35 mm diameter metal ball, a 9.25 mm diameter aluminum rod, and an 11.16 mm diameter aluminum rod… A range of internal eye pressures were produced that varied from 1256 mmHg (24.3 psi) to 22843 mmHg (442 psi)… [and] resulted in zero globe rupture.

Structure of the Eye (With Diagram) | Receptors | Biology

In this article we will discuss about the structure of the eye, with the help of suitable diagrams.

The eye is one of the most important of the receptors. It provides us with information on dimensions, colours and the distance of objects in our environment.

How the Eye Produces a Focused Image:

1. Light rays from an object enter the transparent cornea.

2. The cornea ‘bends’ (refracts) the light rays in towards one another.

3. The light rays pass through the aqueous humour and pupil.

4. The transparent, elastic lens is altered in shape.

i. Fatter, to decrease its focal length, or

ii. Thinner, to increase its focal length.

This is called accommodation.

5. The relatively small amount of refraction now produced by the lens brings the rays to focus on the retina.

6. The retina contains light-sensitive cells:

(i) RODS which work well when light intensity is low, and

(ii) CONES which detect colour.

These cells are stimulated by the light of the image, and convert the light energy into electrical energy.

7. Electrical energy, in the form of an impulse, travels along the optic nerve to the brain.

8. The brain de-codes the impulse to produce the sensation of sight.

Other Important Facts:

(i) The image of objects that we are looking directly at (i.e. which are in the centre of our field of vision) falls on a very sensitive part of the retina called the fovea, or yellow spot. This region has far more cones than rods. Cones provide a picture with greater detail and in better colour.

(ii) There are no rods or cones at the point where the retina is joined to the optic nerve. Images formed on this part of the retina are not converted into impulses and relayed to the brain. This region is called the blind spot. We have blind spots in both of our eyes, but are not usually aware of them. Each eye records a different part of our field of view and covers the blind spot of the other.

The ability of the lens to change shape and focus on objects at different distances is called accommodation.

This ability depends on:

(i) The elasticity of the lens

(ii) The existence of ciliary muscles which are used to alter the shape of the lens

(iii) The suspensory ligaments which transfer the effect of the ciliary muscles to the lens.

The Value of having Two Eyes:

Apart from overcoming the effect of the blind spot, two eyes view the same picture from two slightly different positions. This provides vision in three dimensions, the ability to judge distance (and therefore speed), and offers animals a chance of survival even if one eye is damaged.

The ‘Pupil’ (or Iris) Reflex:

Bright light could seriously damage the delicate light-sensitive cells of the retina. The intensity of light falling on the retina is therefore controlled by the iris. It has an antagonistic arrangement of circular and radial muscles.


This development is an example of sequential inductions where the organ is formed from three different tissues:

Neural tube ectoderm (neuroectoderm) Edit

First, there is an outpocketing of the neural tube called optic vesicles. Development of the optic vesicles starts in the 3-week embryo, from a progressively deepening groove in the neural plate called the optic sulcus. Some studies suggest this mechanism is regulated by RX/RAX transcription factor. [12] The proteins Wnt and FGF (fibroblast growth factor) play a part in this early stage and are regulated by another protein called Shisa. [8] As this expands, the rostral neuropore (the exit of the brain cavity out of the embryo) closes and the optic sulcus and the neural plate becomes the optic vesicle. [13] Optic nerves arise from connections of the vesicles to the forebrain. [1]

Neuroectoderm gives rise to the following compartments of the eye:

Surface ectoderm Edit

Lens development is closely related to optic vesicle development. The interaction between the growing vesicle and the ectoderm causes the ectoderm to thicken at that point. This thickened portion of the ectoderm is called the lens placode. Next, the placode invaginates and forms a pouch referred to as the lens pit. [1] [14] [15] Scientists are studying the tension forces necessary for invagination of the lens placode and current research suggests that microfilaments might be present in early retinal cells to allow for invagination behavior. Research has also shown that Rho GTPase dependent filopodia from the precursor lens ectoderm play an important role in the formation of the lens pit. [16] [17] [18] Eventually, the pit becomes completely enclosed. This enclosed structure is the lens vesicle. [1] Studies have shown that lens development requires the presence of the Pax6 gene, which is the master regulatory gene for eye morphogenesis. [19] This master regulatory gene is not necessary for the closely associated optic vesicle development. [20] Additionally, Ras activation has been shown to be sufficient for starting lens differentiation, but not enough for its completion. [19]

The optic vesicles then begin to form the optic cup [21] [22] . Optic cup morphogenesis is the invagination process occurring after neuroectoderm movement forms the spherical optic vesicle (Phase 1). Invagination is when a tissue folds back on itself. Over the course of approximately 12 hours, the distal end of the optic vesicle inner layer begins to flatten (Phase 2). Over the following 18 hours, both the inner and outer layers begin to flex inward at sharp angles, beginning the formation of a C-shaped edge (Phase 3). The final 18 hours involve continuing this apically convex invagination to form the optic cup [23] [24] . At this point, morphologies such as columnar epithelial cells, pseudo-stratified cells, and apically narrow wedge-shaped cells can be observed. [25]

The inner layer of the optic cup is made of neuroepithelium (neural retina), while the outer layer is composed of retinal pigment epithelium (RPE). Experiments have determined that RPE cell differentiation and maintenance requires interaction with neighboring tissues, most likely canonical Wnt signaling, while neural retina differentiation is driven by tissue-autonomous factors. [25]

Bone morphogenic proteins (BMPs) are important regulators of optic cup development. In fact, research studies have shown that BMP agonists and antagonists are necessary for precision of optic cup development. [20] Interactions between tissues and signaling pathways also play a major role in morphogenesis of the optic cup. [12]

It is of interest to note that research has shown isolating the optic cup from neighboring tissue after completed invagination in tissue culture medium can lead to the development of most major parts of the eye, including photoreceptors, ganglion cells, bipolar cells, horizontal cells, amacrine cells and Muller glia. This indicates that morphogenesis of the optic cup occurs independently of external cues from its environment, including presence of lens. [25] However, the lens is necessary to act as an inducer for the ectoderm to transform it into the cornea.

Surface ectoderm produces the following parts:

Neural crest Edit

Neural crest cells are themselves derived from the ectoderm and lie close to the neural tube:

Mesoderm Edit

Mesoderm contributes to the following structures:

According to Liem et al., the organogenesis of the eye is pointed out as an example of a developmental cascade of inductions. The eye is essentially a derivative of the ectoderm from the somatic ectoderm and neural tube, with a succession of inductions by the chordamesoderm.

Chordamesoderm induces the anterior portion of the neural tube to form the precursors of the synapomorphic tripartite brain of vertebrates, and it will form a bulge called the diencephalon. Further induction by the chordamesoderm will form a protrusion: the optic vesicle. This vesicle will be subsequently invaginated by means of further inductions from the chordamesoderm. The optic vesicle will then induce the ectoderm that thickens (lens placode) and further invaginates to a point that detaches from the ectoderm and forms a neurogenic placode by itself. The lens placode is affected by the chordamesoderm making it invaginate and forms the optic cup composed by an inner layer of the neural retina and outer layer of the pigmented retina that will unite and form the optic stalk. The pigmented retina is formed by rods and cones and composed of small cilia typical of the ependymal epithelium of the neural tube. Some cells in the lens vesicle will be fated to form the cornea and the lens vesicle will develop completely to form the definitive lens. Iris is formed from the optic cup cells.

Only the epidermis in the head is competent to respond to the signal from the optic vesicles. Both the optic vesicle and the head epidermis are required for eye development. The competence of the head epidermis to respond to the optic vesicle signals comes from the expression of Pax6 in the epidermis. Pax6 is necessary and sufficient for eye induction. This competence is acquired gradually during gastrulation and neurulation from interactions with the endoderm, mesoderm, and neural plate.

Sonic hedgehog reduces the expression of Pax6. When Shh is inhibited during development, the domain of expression for Pax6 is expanded and the eyes fail to separate causing cyclopia. [26] Overexpression of Shh causes a loss of eye structures.

Retinoic acid generated from vitamin A in the retina plays an essential role in eye development as a secreted paracrine signal which restricts invasion of perioptic mesenchyme around the optic cup. [27] Vitamin A deficiency during embryogenesis results in anterior segment defects (particularly cornea and eyelids) that lead to vision loss or blindness.

There is some evidence that LMX1B plays a role in periocular mesenchymal survival. [28]

The rods and cones are the site of transduction of light to a neural signal. Both rods and cones contain photopigments. In vertebrates, the main photopigment, rhodopsin, has two main parts Figure 17.19): an opsin, which is a membrane protein (in the form of a cluster of α-helices that span the membrane), and retinal—a molecule that absorbs light. When light hits a photoreceptor, it causes a shape change in the retinal, altering its structure from a bent ( cis ) form of the molecule to its linear ( trans ) isomer. This isomerization of retinal activates the rhodopsin, starting a cascade of events that ends with the closing of Na + channels in the membrane of the photoreceptor. Thus, unlike most other sensory neurons (which become depolarized by exposure to a stimulus) visual receptors become hyperpolarized and thus driven away from threshold (Figure 17.20).

Figure 17.19. (a) Rhodopsin, the photoreceptor in vertebrates, has two parts: the trans-membrane protein opsin, and retinal. When light strikes retinal, it changes shape from (b) a cis to a trans form. The signal is passed to a G-protein called transducin, triggering a series of downstream events.

Figure 17.20.
When light strikes rhodopsin, the G-protein transducin is activated, which in turn activates phosphodiesterase. Phosphodiesterase converts cGMP to GMP, thereby closing sodium channels. As a result, the membrane becomes hyperpolarized. The hyperpolarized membrane does not release glutamate to the bipolar cell.

Scientists discover new layer of the human cornea

Scientists at The University of Nottingham have discovered a previously undetected layer in the cornea, the clear window at the front of the human eye.

The breakthrough, announced in a study published in the academic journal Ophthalmology, could help surgeons to dramatically improve outcomes for patients undergoing corneal grafts and transplants.

The new layer has been dubbed the Dua's Layer after the academic Professor Harminder Dua who discovered it.

Professor Dua, Professor of Ophthalmology and Visual Sciences, said: "This is a major discovery that will mean that ophthalmology textbooks will literally need to be re-written. Having identified this new and distinct layer deep in the tissue of the cornea, we can now exploit its presence to make operations much safer and simpler for patients.

"From a clinical perspective, there are many diseases that affect the back of the cornea which clinicians across the world are already beginning to relate to the presence, absence or tear in this layer."

The human cornea is the clear protective lens on the front of the eye through which light enters the eye. Scientists previously believed the cornea to be composed of five layers, from front to back, the corneal epithelium, Bowman's layer, the corneal stroma, Descemet's membrane and the corneal endothelium.

The new layer that has been discovered is located at the back of the cornea between the corneal stroma and Descemet's membrane. Although it is just 15 microns thick -- the entire cornea is around 550 microns thick or 0.5mm -- it is incredibly tough and is strong enough to be able to withstand one and a half to two bars of pressure.

The scientists proved the existence of the layer by simulating human corneal transplants and grafts on eyes donated for research purposes to eye banks located in Bristol and Manchester.

During this surgery, tiny bubbles of air were injected into the cornea to gently separate the different layers. The scientists then subjected the separated layers to electron microscopy, allowing them to study them at many thousand times their actual size.

Understanding the properties and location of the new Dua's layer could help surgeons to better identify where in the cornea these bubbles are occurring and take appropriate measures during the operation. If they are able to inject a bubble next to the Dua's layer, its strength means that it is less prone to tearing, meaning a better outcome for the patient.

The discovery will have an impact on advancing understanding of a number of diseases of the cornea, including acute hydrops, Descematocele and pre-Descemet's dystrophies.

The scientists now believe that corneal hydrops, a bulging of the cornea caused by fluid build up that occurs in patients with keratoconus (conical deformity of the cornea), is caused by a tear in the Dua layer, through which water from inside the eye rushes in and causes waterlogging.

Endothelium in health

The monolayer of cells called the corneal endothelium that lines the posterior corneal surface is derived from the neural crest during embryologic development. 1 Human endothelial cell density is approximately 6000 cells/mm 2 during the first month of life, 2 but decreases to about 3500 cells/mm 2 by age 5 years. 3 Growth of the cornea accounts for some of this decrease in density, but a decrease in the number of cells also occurs. 2 There is no evidence that human endothelial cells divide under normal circumstances, although they can be induced to divide in cultured corneas. 4,5 They are arrested in the G1 phase of the cell cycle. 6 The reason for the lack of progression into the cell cycle is still unknown, but it may involve cellular contact inhibition. 7 Central endothelial cell density decreases throughout life at an average rate of about 0.6%/year 8 so that the mean cell density was found to decrease from 3400 cells/mm 2 at age 15 to 2300 cells/mm 2 at age 85 years. 9 Racial or geographic differences appear to exist higher cell densities were found in Japanese than in American subjects. 10

The corneal endothelium secretes collagen that forms a basement membrane called Descemet's membrane. At the time of birth, Descemet's membrane is approximately 3 μm thick and consists of collagen in a banded pattern with a periodicity of approximately 110 nm. 11 Throughout life, endothelial cells continue to secrete Descemet's membrane, but the collagen added after birth is not banded. By age 70 years, the average Descemet's membrane is 13 μm thick, including the original 3 μm of anterior banded (foetal) Descemet's membrane and an additional 10 μm of posterior nonbanded Descemet's membrane. After age 70 years, the posterior nonbanded layer is thicker in females than in males. 11 When the endothelial cells are stressed by damage or disease, they may secrete banded Descemet's membrane to form a posterior banded layer. 12 This layer of abnormal Descemet's membrane has also been termed a posterior collagenous layer. 13

The cornea is an exquisite example of natural engineering the requirement for a living, optically clear lens on the surface of the eye was solved by packing collagen and cells in an orderly lamellar arrangement without blood vessels. The crystalline organization and critical spacing of collagen fibrils makes this tissue optically transparent. Any accumulation of fluid would disrupt this spacing and degrade the transparency. The endothelium must serve two functions to maintain the health and clarity of the stroma: it must control hydration (maintain stromal deturgescence) and it must be permeable to nutrients and other molecules from the aqueous humor because these are not supplied by blood vessels as they are in other tissues. This problem was solved by the development of an endothelial layer that behaves as a partial, or leaky, barrier to the movement of fluid and metabolic substrates. The endothelium maintains stromal deturgescence by functioning both as a barrier to fluid movement into the cornea and an active pump that moves ions, and draws water osmotically, from the stroma into the aqueous humor. These cells are metabolically very active, with large numbers of mitochondria, consistent with their need to move water efficiently. Endothelial cells have an incomplete zonula occludens, a leaky tight junction between adjacent cells, and this accounts for the weak endothelial barrier function that allows nutrients and other molecules to enter the stroma. The combined leaky barrier and fluid pump has sometimes been called a pump-leak mechanism. 14 The barrier and pump functions can be measured clinically by fluorophotometry and pachometry. The barrier function can be estimated from the endothelial permeability to fluorescein. 15 The efficiency of the pump can be studied by measuring the corneal deswelling rate from a 10% increase in thickness induced by 2 h of closed-eye aphakic soft contact lens wear. 16 The endothelial pump rate can be calculated from the deswelling rate and the endothelial permeability. 17

Eyes & Vision Science Projects

Eye Chart Vision Test

A Snellen eye chart is used to determine how “normal” your vision is. It sets a standard for what most people should be able to see when they stand 20 feet away from the chart.

20/20 vision just means that when you stand 20 feet away from a Snellen eye chart, you see what a normal human being can see.

If you see 20/40, that means that when you stand 20 feet away from the chart, you see what a normal person sees standing 40 feet away from it. The higher the second number, the worse your vision is. 20/200 (you see at 20 feet what a normal person sees at 200) is the number for legal blindness in the United States.

20/20 vision isn’t perfect, it’s just “normal.” You can have better vision than 20/20. If you have 20/10 you see at 20 feet what most people see at 10. Some animals, like hawks, might have 20/2 vision!

You can use our Snellen eye chart* to compare vision within your family or with your friends.

(This will only give you an approximate idea of your vision. Your optometrist has much more precise tools to find out exactly how well you can see.)

Each line of the chart is labeled on the left side. The second to last line is 20/20.

Tape the eye chart to a wall, making sure it is in plenty of light. Stand twenty feet away from the chart and begin reading each line.

Have a family member or friend watch to see that you are reading each letter correctly. The last line that you are able to read will give you an approximate idea of your vision.

If you can read the very bottom line, your vision is 20/10! Now try covering one eye and just testing the other one. Is one eye better than the other?

Have all of your family members try reading the chart. Do some of you have better vision than others? If you wear glasses, what is your vision with them on and what is it without them?

*Instructions for downloading: The Snellen Eye Chart PDF is 11″ x 17″, so to print correctly you will need to set your print options to “tile.” Printer choices will vary, but you should do something similar to this. Open the PDF and choose Print. Under the page scaling options, select “tile all pages.” This should print the chart on four sheets of paper. You will need to trim the edges so the pieces match up, and then tape or glue them together.

(You can also order an already-printed 11″ x 17″ copy of our Snellen Eye Chart.)

Blind Spot Experiments

The spot where your optic nerve connects to your retina is called the optic disc. There are no photoreceptor cells on this disc, so when an image hits that part of your retina, you can’t see it.

This is your blind spot. You don’t notice this blind spot in every-day life, because your two eyes work together to cover it up.

To find it, draw a filled-in, 1/4″-sized square and a circle three or four inches apart on a piece of white paper.

Hold the paper at arm’s length and close your left eye. Focus on the square with your right eye, and slowly move the paper toward you. When the circle reaches your blind spot, it will disappear!

Try again to find the blind spot for your other eye. Close your right eye and focus on the circle with your left eye. Move the paper until the square disappears.

What happened when the circle disappeared? Did you see nothing where the circle had been?

No, when the circle disappeared, you saw a plain white background that matched the rest of the sheet of paper.

This is because your brain “filled in” for the blind spot – your eye didn’t send any information about that part of the paper, so the brain just made the “hole” match the rest.

Try the experiment again on a piece of colored paper. When the circle disappears, the brain will fill in whatever color matches the rest of the paper.

The brain doesn’t just match colored backgrounds. It can also make other changes to what you see. Try drawing two filled-in rectangles side by side with a circle in between them. A few inches to the right of this, draw a square.

Close your right eye and focus your left eye on the square. Move the paper until the circle disappears and the two separated bars become one bar.

How did that happen? The circle in between the bars fell on your blind spot. When it disappeared, the brain filled in for the missing information by connecting the two bars!

Here is one final experiment with your blind spot. In this instance the brain doesn’t match the blind spot with its immediate white background, but instead with the pattern surrounding it.

Draw a line down the center of your page. On one side draw a small square and on the other draw rows of circles. Color the center circle red and all the others blue.

Close your left eye and look at the square with your right eye. As you move the paper, the red circle should disappear and be replaced by a blue one!

Technology: Improving Eyesight

The general design of the human eye is practically flawless – but each individual eye isn’t.

If you are using contacts or glasses to read this article, you know that your eyes aren’t perfect.

Perhaps you are nearsighted and can’t see objects that are far away very well.

Or maybe you are farsighted and have trouble seeing things close-up. Both of these conditions occur because of the shape of the eyeball.

If your eyeball is too short, the light rays will focus the image behind your retina, instead of on it. This produces farsightedness. If your eyeball is too long, the light rays focus the image in front of the retina, making you nearsighted.

The technology of vision correction has developed over centuries.

The first known eyeglasses were made in the 13th century out of quartz set into bone, metal, or leather.

Eventually the technology for glass-blowing allowed a fine enough quality of glass to be used for lenses.

The biggest problem with these early glasses was keeping them on. It took almost 400 years before someone developed the side arms to rest on the ears!

Most people bought ready-made glasses that would have helped their vision without correcting it precisely.

For example, Benjamin Franklin had two pair of glasses, one for near and one for far. He got tired of changing them, so he cut the lenses in half and repositioned them so that he could see both near and far using the same glasses – the first bifocals!

With the advance of technology, vision-testing equipment has become more and more precise.

Now to obtain a pair of glasses, you must go to an optometrist who will determine exactly what type and strength of lenses you need.

Concave lenses are used for nearsightedness because they bend light away from the center – this stops the light from focusing too far in front of the retina.

Convex lenses are used for farsightedness because they bend light toward the center, causing the light to focus sooner so the image is not focused behind your retina.

Lenses can also be made that will correct other problems in the eye, such as astigmatism, which is an irregular curvature of the cornea.

Contact lenses are a popular alternative to eyeglasses. These lenses fit directly on the cornea, where they “float” on a layer of tears.

They were under experimentation as early as the mid-19th century, though quality and comfort left much to be desired. Now millions of people in the United States use either soft or hard lenses.

Soft contact lenses are made of flexible, water-absorbing plastics. They are more comfortable to wear than hard lenses, which are made of more rigid plastic that does not form to the eye as well. Hard lenses, on the other hand, produce a sharper image.

Some people want a more permanent solution to their vision problems. In recent years, procedures such as LASIK (laser-assisted in-situ keratomileusis) surgery have been developed to remove the need for external lenses like glasses and contacts.

While external lenses change how the light is bent so that it focuses on your retina, laser surgery reshapes the cornea itself.

The process involves a tightly focused beam of ultraviolet light, called an excimer laser. The surgeon first uses a sharp scalpel to cut a flap in the top layer of the cornea, then directs the laser into the middle layer.

As the laser pulses onto this surface, it vaporizes a microscopic portion of the cornea. By controlling the number and location of the pulses, the surgeon controls how much of the cornea is removed.

Noteworthy Scientist: Charles Bell (1774-1842)

Do you ever wonder how great artists can paint a human face that looks perfectly realistic? One of Charles Bell’s contributions to art was an anatomy textbook especially for artists, called Essays on the Anatomy of Expression in Painting.

Charles Bell was an artist himself, as well as a surgeon and anatomist. He was born in Edinburgh, Scotland, the son of a Church of England minister. His older brother John was a surgeon, author, and teacher of anatomy at the University of Edinburgh.

Studying with his brother, Bell developed both his artistic talent and his medical knowledge. After he graduated from the University with a degree in medicine, Bell assisted in teaching his brother’s anatomy class and publishing a four-volume Anatomy textbook.

Eventually Bell moved to London where he did extensive research on nerves, wrote many books and treatises, opened a school of anatomy, and worked as a surgeon.

In 1815 he cared for the wounded after the bloody battle of Waterloo, his skill in surgery holding him in good stead.

His battlefield experience led him to create illustrations of gunshot wounds to be used by surgeons.

Bell’s research on the brain and nerves proved foundational for modern neurology. He determined that nerves only sent information one way: some took sensory information to the brain, and some took commands from the brain to the rest of the body. He also traced nerves from special sensory organs (such as the eye) to specific parts of the brain.

Through all his research and medical illustration, Bell recognized the hand of a Creator. In 1836 he was invited to contribute to a collection of works “On the Power, Wisdom, and Goodness of God as Manifested in the Creation.”

He agreed, and wrote a treatise called The Hand its Mechanism and Vital Endowment, as Evincing Design.

Bell was knighted by King William IV in 1831, and in 1835 he accepted a position as professor of surgery and returned to Scotland.

What is the strength of human cornea? - Biology

The photoreceptive cells of the eye, where transduction of light to nervous impulses occurs, are located in the retina (shown in Figure 1) on the inner surface of the back of the eye. But light does not impinge on the retina unaltered. It passes through other layers that process it so that it can be interpreted by the retina (Figure 1b). The cornea, the front transparent layer of the eye, and the crystalline lens, a transparent convex structure behind the cornea, both refract (bend) light to focus the image on the retina. The iris, which is conspicuous as the colored part of the eye, is a circular muscular ring lying between the lens and cornea that regulates the amount of light entering the eye. In conditions of high ambient light, the iris contracts, reducing the size of the pupil at its center. In conditions of low light, the iris relaxes and the pupil enlarges.

Figure 1. (a) The human eye is shown in cross section. (b) A blowup shows the layers of the retina.

Practice Question

Which of the following statements about the human eye is false?

  1. Rods detect color, while cones detect only shades of gray.
  2. When light enters the retina, it passes the ganglion cells and bipolar cells before reaching photoreceptors at the rear of the eye.
  3. The iris adjusts the amount of light coming into the eye.
  4. The cornea is a protective layer on the front of the eye.

Figure 2. Rods and cones are photoreceptors in the retina. Rods respond in low light and can detect only shades of gray. Cones respond in intense light and are responsible for color vision. (credit: modification of work by Piotr Sliwa)

The main function of the lens is to focus light on the retina and fovea centralis. The lens is dynamic, focusing and re-focusing light as the eye rests on near and far objects in the visual field. The lens is operated by muscles that stretch it flat or allow it to thicken, changing the focal length of light coming through it to focus it sharply on the retina. With age comes the loss of the flexibility of the lens, and a form of farsightedness called presbyopia results. Presbyopia occurs because the image focuses behind the retina. Presbyopia is a deficit similar to a different type of farsightedness called hyperopia caused by an eyeball that is too short. For both defects, images in the distance are clear but images nearby are blurry. Myopia (nearsightedness) occurs when an eyeball is elongated and the image focus falls in front of the retina. In this case, images in the distance are blurry but images nearby are clear.

There are two types of photoreceptors in the retina: rods and cones, named for their general appearance as illustrated in Figure 2. Rods are strongly photosensitive and are located in the outer edges of the retina. They detect dim light and are used primarily for peripheral and nighttime vision. Cones are weakly photosensitive and are located near the center of the retina. They respond to bright light, and their primary role is in daytime, color vision.

The fovea is the region in the center back of the eye that is responsible for acute vision. The fovea has a high density of cones. When you bring your gaze to an object to examine it intently in bright light, the eyes orient so that the object’s image falls on the fovea. However, when looking at a star in the night sky or other object in dim light, the object can be better viewed by the peripheral vision because it is the rods at the edges of the retina, rather than the cones at the center, that operate better in low light. In humans, cones far outnumber rods in the fovea.

Scientists discover new layer of the human cornea

Scientists at The University of Nottingham have discovered a previously undetected layer in the cornea, the clear window at the front of the human eye.

The breakthrough, announced in a study published in the academic journal Ophthalmology, could help surgeons to dramatically improve outcomes for patients undergoing corneal grafts and transplants.

The new layer has been dubbed the Dua's Layer after the academic Professor Harminder Dua who discovered it.

Professor Dua, Professor of Ophthalmology and Visual Sciences, said: "This is a major discovery that will mean that ophthalmology textbooks will literally need to be re-written. Having identified this new and distinct layer deep in the tissue of the cornea, we can now exploit its presence to make operations much safer and simpler for patients.

"From a clinical perspective, there are many diseases that affect the back of the cornea which clinicians across the world are already beginning to relate to the presence, absence or tear in this layer."

The human cornea is the clear protective lens on the front of the eye through which light enters the eye. Scientists previously believed the cornea to be comprised of five layers, from front to back, the corneal epithelium, Bowman's layer, the corneal stroma, Descemet's membrane and the corneal endothelium.

The new layer that has been discovered is located at the back of the cornea between the corneal stroma and Descemet's membrane. Although it is just 15 microns thick—the entire cornea is around 550 microns thick or 0.5mm—it is incredibly tough and is strong enough to be able to withstand one and a half to two bars of pressure.

The scientists proved the existence of the layer by simulating human corneal transplants and grafts on eyes donated for research purposes to eye banks located in Bristol and Manchester.

During this surgery, tiny bubbles of air were injected into the cornea to gently separate the different layers. The scientists then subjected the separated layers to electron microscopy, allowing them to study them at many thousand times their actual size.

Understanding the properties and location of the new Dua's layer could help surgeons to better identify where in the cornea these bubbles are occurring and take appropriate measures during the operation. If they are able to inject a bubble next to the Dua's layer, its strength means that it is less prone to tearing, meaning a better outcome for the patient.

The discovery will have an impact on advancing understanding of a number of diseases of the cornea, including acute hydrops, Descematocele and pre-Descemet's dystrophies.

The scientists now believe that corneal hydrops, a bulging of the cornea caused by fluid build up that occurs in patients with keratoconus (conical deformity of the cornea), is caused by a tear in the Dua layer, through which water from inside the eye rushes in and causes waterlogging.

Limbal epithelial stem cells of the cornea

The cornea on the front surface of the eye is our window to the world, hence maintenance of corneal tissue transparency is essential for vision. The integrity and functionality of the outermost corneal layer, the epithelium, plays a key role in refraction of light on to the retina at the back of the eye. Like other epithelia, the epithelium of the cornea is maintained by stem cells. This review will discuss what is currently known about the properties of these stem cells, the clinical consequences of stem cell failure and the potential for stem cell therapy in regeneration of the ocular surface.

1. Function and structure of the cornea

The cornea is responsible for protecting the eye against insults such as injury and infection. It also provides the majority (two thirds) of the total refractive power of the eye and is therefore the major refracting lens (Meek et al., 2003).

The cornea is comprised of five layers (see Figure 1), the outermost non-keratinised stratified epithelium, Bowman's layer, a highly ordered keratocyte-populated collagenous stroma, Descemet's membrane and the inner endothelium (a cellular monolayer).

At the outer surface of the cornea, there is an epithelial layer, which sits on a basement membrane above Bowman's layer. The middle stromal layer, which is sparsely populated with keratocytes is surrounded by dense connective tissue. The final layer consists of a single sheet of endothelial cells, which sits on Descemet's membrane.

2. Corneal development

Development of the anterior chamber of the eye (comprised of the cornea, lens, ciliary body, iris, trabecular meshwork and aqueous humour) requires the interaction of cells from the surface epithelium and neuroepithelium with mesenchymal cells predominantly of neural crest origin.

Anterior eye development first begins with the formation of the lens placode. This forms after the optic vesicles come in contact with the surface ectoderm. A thickening forms, that enlarges and forms a lens pit. Between days E8.5 and 9.5 in mouse this lens pit becomes the lens vesicle and remains connected to the surface ectoderm via a lens stalk (Kaufman, 1992 Pei and Rhodin, 1970). Eventually this lens vesicle detaches from the surface ectoderm and invaginates into the optic cup. Shortly after this detachment, periocular mesenchymal cells derived from somitomeric mesoderm and forebrain neural crest migrate into the space between the anterior lens vesicle epithelium and the surface ectoderm, eventually forming keratocytes and corneal endothelium (Trainor and Tam, 1995).

In mice, four to seven layers of mesenchymal cells are seen at E12. These cells have long cytoplasmic extensions with a star shaped phenotype (Haustein, 1983). Cell numbers continue to increase and condense to form several layers of separated flattened cells. At E14.5 to E15.5 the cells adjacent to the lens structure form the endothelium (Reneker et al., 2000). The surface ectoderm cells overlaying the mesenchymal cells become the corneal epithelium. The remaining mesenchymal cells between these two layers differentiate into corneal stromal fibroblasts (Cintron et al., 1983). This differs with humans, where there is a second wave of mesenchymal cell migration into the space between the newly formed endothelial layer and the surface ectoderm. These cells differentiate into corneal fibroblasts. In mouse the proliferative potential of corneal fibroblasts diminishes during development from birth to eyelid opening, however they arrest in the G0 phase of the cell cycle as opposed to becoming terminally differentiated (Zieske et al., 2001). As the corneal endothelium differentiates, the lens detaches from the immature corneal structure. This allows the formation of a fluid filled area in to which the iris and ciliary body grow.

Studies resulting in abnormal corneal development due to the over expression of growth factors such as TGFα, FGF3 and EGF in the lens, highlight the importance of the lens in cornea development (Coulombre and Coulombre, 1964 Reneker et al., 1995 Reneker et al., 2000 Robinson et al., 1998). The ectoderm overlaying the lens becomes the corneal epithelium. In its primitive state the epithelium is 1–2 cell layers thick and later stratifies to three to four cell layers following lens detachment. The eyelids then form and fuse with the primitive epithelium being reduced to 1–2 cells layers thick until eyelid opening which occurs at 24 weeks gestation in humans and P12 to P14 in mice.

For up to seven days of age the corneal and limbal epithelia in rats is 1–2 cells layers (Chung et al., 1992). Prior to eyelid opening at 10 days the epithelial thickness increases to two to three layers. Further increases to four-five cell layers occur in the central cornea following eyelid opening at two weeks of age (Chung et al., 1992 Watanabe et al., 1993). The layers continue to increase until four weeks of age when the epithelium reaches adult levels of six to seven cell layers (Song et al., 2003). The basal epithelial cell shape also changes with development. Initially the cells are flat and ovoid in shape until eyelid opening after which they become more cuboidal. By three weeks the basal cells are more columnar in the central cornea but not the limbal region (Chung et al., 1992).

Between days P1 and P7 the epithelial layer is 1-2 cell layers thick until just prior to eyelid opening at day P10, when this increases to 2-3 cell layers. Following eyelid opening at day P14 the number cell layers increases to 4-5 with 5-6 cells layers being present at 3 weeks of age. At P28 the corneal epithelium is representative of the adult epithelium, with a single layer of columnar basal cells, which become flattened as they move to the surface.

3. The stroma and endothelium

The stroma is a mesenchymal tissue derived from the neural crest. The dense tissue of the stroma accounts for 90% of the total corneal thickness. The parallel arrangement of lamellae formed from heterodimeric complexes of type I and type V collagen fibres maintain transparency (Fini and Stramer, 2005). These collagen fibres are held in a uniform spacing pattern by proteoglycans. Keratocytes (fibroblasts) are located between the lamellae (Hay et al., 1979). These sparsely located keratocytes link to one another via dendritic processes (Muller et al., 1995) and produce crystalline proteins to maintain corneal transparency (Jester et al., 1999). Recent reports have described a keratocyte stem cell population in the anterior stroma (Du et al., 2005 Funderburgh et al., 2005).

Descemet's membrane rests on the innermost surface of the cornea. It acts as a basement membrane for the inner endothelial cell monolayer. These cells transport nutrients from the aqueous humour to the stroma and concurrently pump out excess water preventing corneal oedema (swelling) by maintaining optimal hydration.

4. The corneal epithelium

The corneal epithelium is a dynamic physical barrier preventing the entry of deleterious agents into the intraocular space. It consists of superficial squamous cells, central suprabasal cells and a single layer of inner columnar basal cells. The differentiated squamous cells have surface microvilli and occupy the outer 1–3 cell layers of the epithelium. The function of the microvilli is to increase cell surface area allowing close association with the tear film. Highly resistant tight junctions formed between neighbouring cells provide a protective barrier (Klyce, 1972). The underlying suprabasal cells have wing-like extensions, rarely undergo division and migrate superficially to differentiate into squamous cells.

The inner basal cells consist of a single layer of columnar cells with several important functions including the generation of new suprabasal cells. Additionally, they secrete matrix factors important for basement membrane and stromal function. The basal cells also regulate organisation of hemidesmonsomes and focal complexes to maintain attachment to the underlying basement membrane. These functions are suggested to be important in mediating cell migration in response to epithelial injury (Pajoohesh-Ganji and Stepp, 2005).

5. Homeostasis in the corneal epithelium

Corneal integrity and therefore function is dependent upon the self-renewing properties of the corneal epithelium. The prevailing hypothesis is that this renewal relies on a small population of putative stem cells located in the basal region of the limbus. These putative stem cells are primitive and can divide symmetrically to self renew and asymmetrically to produce daughter transit amplifying cells (TAC) that migrate centripetally to populate the basal layer of the corneal epithelium (see Figure 3 Kinoshita et al., 1981 Tseng, 1989). The TAC divide and migrate superficially, progressively becoming more differentiated, eventually becoming post-mitotic terminally differentiated (TD) cells. Using suppressive subtractive hybridisation, Sun et al., 2006 identified a novel gene (EEDA) with localisation to corneal basal and suprabasal cells, suggesting it is involved in early stage stratification of epithelial differentiation (Sun et al., 2006).

Limbal epithelial stem cells reside in the basal layer of the epithelium (Ep), which undulates at the limbus. Daughter transient amplifying cells (TACs) divide and migrate towards the central cornea (arrowed) to replenish the epithelium, which rests on Bowman's layer (BL). The stroma (St) of the limbal epithelial stem cell niche is populated with fibroblasts and melanocytes and also has a blood supply.

Once fully differentiated TD squamous cells are shed from the ocular surface during normal wear and tear and this in turn stimulates the cycle of cell division, migration and differentiation (Beebe and Masters, 1996). Thoft and Friend developed the ‘The X, Y, Z hypothesis of corneal epithelial maintenance’. This hypothesis proposed that the addition of the proliferation of basal cells (X) and the centripetal migration of cells (Y) was equal to epithelial cell loss from the corneal surface. However, they were unable to rule out the involvement of the neighbouring bulbar conjunctiva (Thoft and Friend, 1983). Later, mathematical analysis indicated that the corneal epithelial cell mass could be renewed by cells from the limbal epithelium alone (Sharma and Coles, 1989). Furthermore, a fine balance between cell proliferation, differentiation, migration and apoptosis is necessary. A variety of cytokines have been shown to play important roles in the maintenance and wound healing of the cornea. These factors are supplied in part by the adjacent tear film and the aqueous humour (Welge-Lussen et al., 2001). Other growth factors are produced by keratocytes in the supporting stroma (West-Mays and Dwivedi, 2006) and by the corneal epithelial cells themselves (Rolando and Zierhut, 2001).

6. Limbal epithelial stem cells

Throughout life, our self-renewing tissues rely upon populations of stem cells / progenitors to replenish themselves throughout life following normal wear and tear and injury. The corneal epithelium on the front surface of the eye is no exception as dead squamous cells are constantly sloughed from the corneal epithelium during blinking. At the corneo-scleral junction in an area known as the limbus, there is a population of limbal epithelial stem cells (LESCs). LESCs share common features with other adult somatic stem cells including small size (Romano et al., 2003) and high nuclear to cytoplasmic ratio (Barrandon and Green, 1987). They also lack expression of differentiation markers such as cytokeratins 3 and 12 (Kurpakus et al., 1990 Schermer et al., 1986).

LESCs are slow cycling during homeostasis and therefore retain DNA labels for long time periods, however in the event of injury they can become highly proliferative (Cotsarelis et al., 1989 Lavker and Sun, 2003 Lehrer et al., 1998). To replenish the stem cell pool, stem cells have the ability to divide asymmetrically (see Figure 4).

It is thought LESC undergo asymmetric cell division producing a stem cell, which remains in the stem cell niche to repopulate the stem cell pool, and a daughter early transient amplifying cell (eTAC). This more differentiated eTAC is removed from the stem cell niche and is able to divide further producing transient amplifying cells (TAC), eventually giving rise to terminally differentiated cells (DC). The double arrows represent the self-renewing capability of the stem cells. The supporting niche cells (blue) surround the stem cells (light green).

Expression of C/EBPΔ in a subset of LESC both in vivo and in vitro has recently been suggested to be involved in the regulation of self-renewal and LESC cell cycle length (Barbaro et al., 2007).

7. Evidence for stem cells in the corneal limbus

The first experimental indication of the presence of stem cells in the limbus was the observation of pigment (melanin) movement from the limbus to towards an epithelial defect following wounding of rabbit corneas (Mann, 1944).

Davanger and Evenson later observed a similar centripetal migration of pigment from limbus to central cornea in humans. Hence they proposed that the limbal Palisades of Vogt (PV) were the source of LESC (Davanger and Evenson, 1971 Huang and Tseng, 1991). Following lamellar keratoplasty, this centripetal migration was also observed in the rabbit as host epithelium was gradually replaced with donor epithelium (Kinoshita et al., 1981). Furthermore, the complete removal of the limbus results in impaired corneal function, neovascularisation and conjunctival ingrowth (Huang and Tseng, 1991).

Stem cells may be identified by the retention of DNA labels as they are slow cycling and only divide occasionally (Bickenbach, 1981). Assuming stem cell division during the labelling period, stem cell exposure to DNA precursors such as tritiated thymidine or bromodeoxyuridine followed by chase periods of up to 8 weeks labels the slow cycling cells (presumed to be stem cells). The more differentiated and more rapidly dividing daughter transit amplifying cells (TAC) undergo dilution of the label through multiple divisions. Through the use of tritiated thymidine, Cotsarelis et al, found slow cycling label retaining cells (LRCs) in the limbal basal epithelial region of the mouse cornea and postulated that up to 10% of limbal basal cells were stem cells (Cotsarelis et al., 1989). Phenotypically this population of cells appear to be more primitive in nature as they remain small and round (Romano et al., 2003).

Limbal basal cells exhibit higher proliferative potential when compared to peripheral and central cornea both in vitro and in vivo. Large epithelial wounds in rabbits heal faster than smaller central defects. This implies that the proliferative capacity of the peripheral cornea is greater than that of the central (Lavker et al., 1991). In the human, limbal explant cultures have greater proliferative potential when compared to central explants (Ebato et al., 1987 Ebato et al., 1988). Furthermore, LESC proliferation is resistant to inhibition by tumour-promoting phorbol esters (Kruse and Tseng, 1993 Lavker et al., 1998). Based upon the methods of characterisation used to identify features of stem cells isolated and cultured from human epidermis (Barrandon and Green, 1987), similar clonogenicity studies on cells isolated from the limbus produced large holoclone colonies (stem cell derived) with extended cell generation number. The less clonogenic meroclones and paraclones were found elsewhere in the cornea (Pellegrini et al., 1999).

Clinical evidence also points toward the limbus as a depository for a stem cell population. During homeostasis, the limbal epithelial cells are thought to act as a barrier preventing conjunctival epithelial cells from encroaching upon the cornea (Tseng, 1989). During LESC failure (to be discussed later), the conjunctiva can invade the cornea causing chronic inflammation, painful corneal opacity and neovascularisation. Ambati et al., have recently shown experimentally that soluble vascular endothelial growth factor receptor 1 (sFlt1) is important for corneal avascularity (Ambati et al., 2006). They have since found expression of sFlt1 in normal human corneal epithelium and a reduction of sFlt1 in vascularised patients (Ambati et al., 2007). Further clinical evidence pointing to the location of LESC at the limbus was demonstrated by Kenyon and Tseng, who transplanted two limbal explants taken from the contralateral healthy eye of patients on the damaged eye. This resulted in re-epithelisation of the cornea and regression of persistent epithelial defects and neovascularisation (Kenyon and Tseng, 1989).

The dogma that stem cells which give rise to corneal epithelial cells exclusively reside in the limbus was recently challenged. In the mouse it was demonstrated that central corneal epithelium could be serially transplanted and that it contains oligopotent stem cells that can maintain the corneal epithelium without cellular input from the limbal region. Furthermore, holoclone colonies were cultured from the central corneas of a number of mammalian species including from two human donors (Majo et al., 2008). However, both human donors were 4 years or younger so it will be interesting to see if the results are reproducible in the adult human cornea when development of the eye is complete.

In the skin, the existence of transit amplifying cells has also been questioned. Rather than stem cells producing transit amplifying cells to maintain homeostasis in the epidermis, it has been proposed that a population of ‘committed progenitor’ cells fulfil this function during normal tissue turn over. It is proposed that the stem cells are only called into action in response to injury (Clayton et al., 2007 Jones et al., 2007). Similarly, it has been proposed that function of LESCs is to respond to injury and not to look after normal wear and tear of the corneal epithelium (Majo et al., 2008). It remains to be determined if the long-accepted transit amplifying cell hypothesis continues to hold true for the corneal epithelium.

7.1. The LESC niche

The stem cell niche, or microenvironment consisting of cellular and extracellular components, is hypothesised to prevent stem cell differentiation and thus regulates their fate (Schofield, 1983 Watt and Hogan, 2000). When a stem cell divides asymmetrically, one daughter may leave the niche to enter a differentiation pathway under the influence of different environmental stimuli. The limbus differs from cornea both anatomically and functionally and hence could differentially determine stem cell fate.

Within the limbal region of the cornea, the LESC niche is thought to be located within the palisades of Vogt (PV) – an undulating region of increased surface area. The palisades are highly pigmented with melanocytes (Davanger and Evenson, 1971 Higa et al., 2005) and are infiltrated with Langerhan's cells (Baum, 1970) and T-lymphocytes (Vantrappen et al., 1985). The melanin pigmentation is thought to shield LESCs from damaging ultraviolet light and the resultant generation of reactive oxygen species (Shimmura and Tsubota, 1997). The deep undulations of the Palisades of Vogt at the limbus provide LESC with an environment that protect them from shearing forces (Gipson, 1989). Furthermore the crypts described by Shortt et al., predominantly occur on the superior and inferior cornea where they are normally covered by the eye lids. (Shortt et al., 2007a) This may reflect the evolution of a protective environment for LESCs in humans. The basement membrane lining the LESC niche contains papillae of stroma that project upwards (Shortt et al., 2007a). The limbal and corneal basement membrane components also differ, with the limbal region containing laminin-1,5 and α2β2 chains not found in the cornea. Furthermore, type IV collagen α1, α2 and α5 chains are found in the limbal region whereas α3 and α5 are located in the cornea (Ljubimov et al., 1995 Tuori et al., 1996). A more recent study by Schlötzer-Schrehardt et al., found patchy immunolocalisation of laminin γ3 chain, BM40/SPARC and tenancin C, that was also found to co-localise with ABCG2/p63/ K19 -positive cell clusters. These factors may be involved in retaining cell stemness (Schlotzer-Schrehardt et al., 2007).

The basement membrane beneath the LESC may also act to sequester and therefore modulate growth factors and cytokines involved in LESC regulation and function (Klenkler and Sheardown, 2004). Although the surface of the cornea is exposed to atmospheric oxygen, the LESC niche lies beneath a number of cell layers where the oxygen tension is likely to be lower. Interestingly, hypoxic in vitro conditions have been found to produce larger, less differentiated limbal epithelial cell colonies suggesting that low oxygen levels may induce selective proliferation of undifferentiated cells (Miyashita et al., 2007).

The limbal niche is vascularised and highly innervated (Lawrenson and Ruskell, 1991) unlike the avascular cornea and therefore is a potential source of nutrients and growth factors for LESC. Limbal fibroblasts in the underlying stroma are heterogeneous and express secreted protein acidic and rich in cysteine (SPARC) that may contribute to LESC adhesion (Shimmura et al., 2006). Furthermore, Nakamura et al., identified a population of bone marrow-derived cells located in the limbal stroma following transplantation of GFP labelled bone marrow cells into nude mice (Nakamura et al., 2005). It is possible therefore that these cells are able to migrate into the limbal stroma, although any potential functionality remains unclear.

Sonic hedgehog, Wnt/β-catenin, TGF-β and Notch signalling pathways have all being implicated in niche control of stem cells, however little is known of their potential roles in the LESC niche. Mice lacking in expression of Dkk2 , a Wnt pathway inhibitor, display epidermal differentiation on the ocular surface. The lack of Dkk2 , leads to increased Wnt/β-catenin signalling in the limbal stroma. This demonstrates the importance of limbal niche control over LESC differentiation during development. PAX6 expression is also lost in the corneal epithelial cells of these mice, suggesting it is downstream of Dkk2 (Mukhopadhyay et al., 2006). Deficiencies in PAX6 leads to aniridia resulting in impaired corneal epithelial function and eventual LESC failure, which may be due to altered niche development.

7.2. Putative positive and negative LESC markers

The literature reflects many attempts to prospectively identify LESC using a specific marker. As yet no single, reliable marker has been found. However, the expression of a combination of several features seems to allow for greater specificity.

Putative ‘markers’ can either be positive (present) or negative (absent). Limbal basal cells lack differentiation markers such as the 64 kDa cytokeratin 3 (CK3) that is present in all other layers of the corneal epithelium and the suprabasal layers of the limbal epithelium (Schermer et al., 1986). The corneal specific 55 kD protein, cytokeratin 12 (CK12) is also expressed in a similar pattern (Chaloin-Dufau et al., 1990). Furthermore, connexin 43 (Shortt et al., 2007a Matic et al., 1997) and involucrin (Chen et al., 2004), both markers of cells destined for differentiation, are also absent.

The transcription factor p63 is required for formation of epidermis and has been proposed as a putative positive LESC marker (Pellegrini et al., 2001). In vitro, p63 was found to be expressed in limbal epithelial cell derived holoclones with little or no expression in meroclones and paraclones. In vivo, p63 was located in the limbal basal epithelium. However, since these initial observations a number of reports have suggested that p63 is not sufficiently specific to act as an LESC marker as it has also been localised to basal cells of the peripheral and central cornea in humans (Chen et al., 2004 Dua et al., 2003) and in rats (Chee et al., 2006). However, limbal epithelial cells expressing high levels of p63 with a high nuclear to cytoplasmic ratio appear to be more stem like (Arpitha et al., 2005). Further work has since indicated that the ΔNp63α isoform may more specifically label LESC (Di Iorio et al., 2005).

Many types of organ-specific stem cells, including LESC have been recently shown to exhibit a side population (SP) phenotype. The SP cells are able to efflux Hoechst 33342 dye through the ATP-binding cassette transporter Bcrp1/ ABCG2 . ABCG2 has therefore been proposed to be a universal marker for stem cells (Zhou et al., 2001 Watanabe et al., 2004). In putative LESCs, this protein has been immunolocalised to the cell membrane and cytoplasm of a population of limbal basal cells and a few suprabasal cells (Chen et al., 2004). Furthermore, ABCG2 positive cells produce higher colony forming efficiency values in vitro than their negative counterparts (de Paiva et al., 2005). Our laboratory has localised ABCG2 to the outer edge of holoclones where it is thought that the stem cells reside.

Clusters of cells expressing the integrin α9 have been localised to the limbal basal epithelium (Stepp et al., 1995). However, upregulation of α9 in wounded murine corneas have since indicated this integrin to be associated with TAC's (Stepp and Zhu, 1997). Integrin β1 was originally suggested to be a keratinocyte marker (Jones and Watt, 1993). Cells that rapidly adhere to the integrin β1 ligand, collagen IV also display LESC properties (Li and Lu, 2005). Limbal basal epithelial cells are described as β1 integrin bright as are the stem cells of the epidermis suggesting a gradient of expression that decreases with differentiation. The integrins α2, α6 and β4 are negative in the limbal basal epithelial cells (Schlotzer-Schrehardt and Kruse, 2005).

N-cadherin is an important mediator of cell-cell adhesion and may play a key role in the maintenance of haemopoietic stem cells by facilitating adhesion to osteoblasts in the bone marrow niche (Calvi et al., 2003 Zhang et al., 2003). Hayashi et al found expression of N-cadherin in a subpopulation of limbal epithelial basal cells and in adjacent melancytes implying N-cadherin plays an important role in interactions between LESC and their corresponding niche cells (Hayashi et al., 2007).

Even though the limbal epithelium is derived from the surface ectoderm a number of neural stem cell markers have been suggested as LESC markers. Recent in depth immunological studies of neurotrophic factors and their receptors in the human has found NGF , glial cell-derived neurotrophic factor ( GDNF ) and their corresponding receptors TrkA and GDNF family receptor alpha (GFRα)-1 to be exclusively expressed in the limbus (Qi et al., 2008).

Notch 1 is a ligand-activated transmembrane receptor that has been shown to maintain progenitor cells in a number of tissues. The role of Notch signalling in the cornea is unclear. However, cell clusters in the palisades of Vogt have been found with some co-localisation with ABCG2 (Thomas et al., 2007). Using Notch 1 deficient mice, Vauclair et al, demonstrated Notch 1 signalling is required for cell fate maintenance during corneal epithelial wound healing linking this to regulation of vitamin A metabolism (Vauclair et al., 2007). Notch 1, other Notch family members and their down-stream targets have been identified throughout the cornea suggesting a role in differentiation (Ma et al., 2007). More recently, Nakamura et al., has found Hes1 , a major target in Notch1 signalling, to be localised to the basal limbal epithelium in adult mice (Nakamura et al., 2008). It is likely that Notch signalling, perhaps under synergistic regulation with the Wnt signalling pathway, controls the balance between LESC self-renewal and daughter cell commitment to differentiation. The cell cycle arrest transcription factor C/EBPΔ has also been implicated in the regulation of LESC self-renewal. Limbal epithelial basal cells that express C/EBPΔ co-express Bmi1 (which is involved in stem cell self renewal) and ΔNp63α (Barbaro et al., 2007).

Cell-cell communication is facilitated by gap junctions. Connexins 43 and connexin 50 are present in the corneal epithelium (Dong et al., 1994). Cx 43 is expressed by corneal basal cells except that of the limbus, implying it is utilised by more early TACs. The lack of intracellular communication has been suggested to help maintain stem cells and their niche (Matic et al., 1997) by protecting the cells from damage affecting adjacent neighbours (Chee et al., 2006). Like the stratified squamous epithelia, (Watt and Green, 1981) involucrin is also expressed in the corneal epithelium (Chen et al., 2004) and in larger cells in vitro suggesting it is a marker of differentiation.

The RNA binding protein, Musashi-1 is produced in the developing and adult eye (Raji et al., 2007) and has recently been found in putative LESCs co-cultured with amniotic epithelial cells as feeders (Chen et al., 2007).

7.3. Clinical consequences of LESC failure and cultured stem cell therapy

An alkali burn to the human cornea can cause ocular surface failure with neovascularisation, opacification and blindness resulting from LESC deficiency.

The biological mechanisms of efficacy experienced by recipients of the cultured LESCs are unclear yet the clinical results are promising. It has been suggested that bone marrow derived stem cells may be recruited to the cornea to repair the damage caused by LESC failure (Daya et al., 2005) since no long-term survival of allogeneic cultured LESCs has been demonstrated. Our hypothesis is that the transplanted cultured limbal epithelium may act, at least in some patients, by ‘kick-starting’ the recipient's own ailing LESC.

One of the causes of blindness in children with aniridia is due to progressive ocular surface failure. The majority of cases are caused by PAX6 haploinsufficiency being a result of heterozygous null mutations (Van Heyningen and Williamson, 2002). The disease is a pan-ocular, bilateral condition most prominently characterised by iris hypoplasia and varies from a relatively normal iris to the complete lack of an iris. Aniridia is often associated with cataracts, corneal vascularisation and glaucoma, with a significant number of cases of visual morbidity being due to corneal abnormalities. The underlying process of these abnormalities is poorly understood and is thought to be due to stem cell failure (Mackman et al., 1979 Nishida et al., 1995 Tseng and Li, 1996). However, it has also been proposed that it may be due to a deficiency in the stem cell niche and adjacent corneal stroma (Ramaesh et al., 2005). More recently downregulation of Pax6 has been linked to abnormal epidermal differentiation of cornea epithelial cells (Li et al., 2008). Treatment usually involves replacement of LESC using limbal allografts and/or corneal grafts or more recently ex vivo cultured LESC grafts (Holland et al., 2003).

Aniridia represents a spectrum of disease, with iris anatomy defects ranging from the total absence of the iris to mild stomal hypoplasia with a pupil of normal appearance. Other associated defects include foveal hypoplasia, optic nerve hypoplasia, nystagmus, glaucoma and cataracts. These conditions may develop with age causing progressive visual loss. Another important factor leading to progressive loss of vision is aniridic-related keratopathy (ARK Mackman et al., 1979 Margo, 1983) which occurs in 90% of patients. Initially the cornea of patients appears normal during childhood (Nelson et al., 1984 Nishida et al., 1995). Changes occur in patients in their early teenage years, with the disease manifesting as a thickened irregular peripheral epithelium. This is followed by superficial neovascularisation and if left untreated it may result in subepithelial fibrosis and stromal scarring. Furthermore patients develop recurrent erosions, ulcerations, chronic pain and eventual blindness (Holland et al., 2003). Histologically, stromal neovascularisation and infiltration of inflammatory cells is seen with the destruction of Bowman's layer. Additionally, the presence of goblet and conjunctival cells is seen on the corneal surface (Margo, 1983). Traditionally, these clinical and histological manifestations have lead to the consensus that LESC deficiency is largely responsible for corneal abnormalities in aniridia (Dua and Azuara-Blanco, 2000 Margo, 1983).

Based on the clinical and histological manifestation of aniridia, LESC deficiency has been presumed to be the pathogenesis behind ARK (Dua et al., 2000 Margo, 1983 Nishida et al., 1995). As a LESC marker has yet to be definitively identified, a true demonstration of LESC deficiency can not be assumed. Furthermore treatment for these patients involving replacement of LESC, either by keratolimbal allografts or more recently ex vivo expanded LESC grafts, provides a better outcome than corneal transplants (Holland et al., 2003 Shortt et al., 2007b Tiller et al., 2003). This is consistent with LESC deficiency. However, patients who receive both limbal and corneal tissue seem to have the better outcome, suggesting an abnormality with corneal tissue and not just the limbus. This may be a downstream effect of LESC deficiency. Alternatively low levels of PAX6 may have a generalised effect on the entire cornea. ARK could also be the consequence of abnormal corneal epithelial/stromal healing responses as there is insufficient evidence to indicate that the proliferative potential of LESC is impaired (Ramaesh et al., 2005 Sivak et al., 2000). Recently, studies looking at the regulation of genes downstream of Pax6 in the Pax6 heterozygous mouse, suggests the pathogenesis of ARK is due a number of mechanisms and not solely due to LESC deficiency (Ramaesh et al., 2005). Further studies are needed to elucidate the exact mechanism of ARK progression to allow the use of appropriate treatments.

Mutations in Pax6 result in a distinct small eye syndrome in the small eye (SEY) mouse and rat (Hill et al., 1991 Matsuo et al., 1993). These animals are excellent models for aniridia and the progressive nature of associated corneal abnormalities (Ramaesh et al., 2003 Davis et al., 2003). As the name suggests, mice with semidominant mutations develop small eyes and other ocular deformities. The murine strains Pax6 Sey , Pax6 SeyNeu and Pax6 Coop represent three SEY mice with differing point mutations in the Pax6 gene. Pax6 SeyDey and Pax6 SeyH mice have Pax6 gene deletions (Hill et al., 1991 Hogan et al., 1986 Lyon et al., 2000 Schmahl et al., 1993 Theiler et al., 1980). The SEY mice with semidominant heterozygous phenotypes demonstrate comparable developmental ocular abnormalities. This includes microophthalmia and defects in the iris, lens and retina with phenotypic severity being variable (Callearts et al., 1997 Hill et al., 1991). Cataracts, glaucoma and more importantly corneal abnormalities can develop in mutant SEY during post-natal development and adult life (Lyon et al., 2000). Interestingly, the phenotypic variability seen between mice is also observed within a single SEY strain (Hogan et al., 1986). This can even be detected between two eyes of the same mouse, suggesting a stringent requirement for Pax6 activity to be at specific levels at precise times during development (Hill et al., 1991 Schedl et al., 1996 van Ramsdonk and Tilghman, 2000). Homozygotes generate an ultimately lethal phenotype with no eyes and nasal primordial (Hill et al., 1991). A number of sey mice arose independently all of which are semidominant and by examining comparative mapping studies and phenotypic similarities to aniridia, it was suggested to be the mouse homologue of the human disease (Glaser T et al., 1990). This research led to the discovery that the Pax6 gene was responsible for the Sey phenotype and suggested that it was also responsible for the human disease, aniridia (Hill et al., 1991). These models are helping us to address fundamental questions about LESCs and their niche environment.

8. Summary

LESCs are clearly important for vision. Efforts to specifically and prospectively identify these elusive cells are proving difficult. However, despite this mixed populations of epithelial cells isolated from the limbal region have the potential to restore the ocular surface and improve vision in patients with LESC function failure. The mode of clinical efficacy (and treatment failure) may become apparent once a more thorough understanding of normal LESC regulation and the role of the niche is gained.