Ординатура / Офтальмология / Английские материалы / Corneal Endothelial Transplant (DSAEK, DMEK & DLEK)_John_2010

the unwanted reflected light into a light sink, thus eliminating the reflected light interference prevalent in prior designs.1 However, this apparent simple solution required a complex arrangement of additional lenses and prisms within the optical system to bend the light rays to ensure that they correctly passed through the Nipkow disk’s pinholes.1 Overall, the end-result of these modifications was a moderate decrease in image quality secondary to induced astigmatism. In addition, a miscalculation in the location of the reflected ray’s focal point as small as 1 mm decreased confocality.1

A simplified light path using a single-sided confocal design was designed to eliminate the complications of Kino’s design, while maintaining the reliability and simplified alignment characteristics of the single-sided disk system. Although this new single-sided confocal microscopy system has been produced as a prototype, it is not commercially available.

Scanning-Slit Confocal Microscope

Koester invented a different confocal technique that substituted a slit-beam for the Nipkow disk. He modified a specular microscope by adding a scanning-slit and mirror system1, 25 Maurice had also developed a similar system.1,14 Both designs used a novel approach without using the Nipkow pinhole disk. This design substituted a fine slitbeam of light. To allow for scanning, Koester and Maurice used movable mirrors to bend the light rays, allowing for X-Y plane scanning.1 They then combined these movable mirrors with lenses to create a mechano-optical scanning mirror apparatus.1 To eliminate aberrant rays, the reflected light rays passed through an additional slit before reaching the detector similar to the tandem confocal concepts.1 Like the pinhole effect, this scanning slit-beam design only imaged the focal plane of interest and allowed for dynamic, three-dimensional scanning.

However, this microscope possessed a less precise Z- axis resolution.15 Early designs were plagued with slow scan times as compared to the tandem scanning technique. This limitation made it difficult to reach the 30 images per second scan speed threshold, which is required for full resolution video image capture.1 However, new low-lux detectors (cameras) and new, more powerful illuminators have helped to overcome these limitations.1

Laser Scanning Confocal

Microscope

Laser Scanning Confocal Microscopy (LSCM) is a relatively new confocal technique that has found wide applications

in the biological sciences. Developed by the MRC Laboratory of Molecular Biology in Cambridge, UK in 1986, this design has evolved into a highly successful commercial product.12 The rapid utilization of this technique is easily explained by the wide use of fluorescent antibodies in biological research.

Although the fluorescent-labeled antibodies were introduced in 1941, their true significance in identifying biological structures was not appreciated until the 1970’s when researchers realized that these antibodies could be used to tag biological proteins, such as actin and tublin.12,16 After this discovery, the florescent microscope quickly dominated biological research.12 However, researchers quickly grew frustrated with the aberrant light scattering inherent when examining thicker tissue specimens. To resolve this issue, they investigated and adapted Minsky’s concept of confocal microscopy.

However, there were significant differences in design. The Nipkow disk design was abandoned since the pinholes did not allow enough light to pass through the system to make this system practical.12 After years of research, the modern laser scanning confocal microscope emerged. Currently, there are numerous design variations, but many of these designs encompass the same concept. Modern designs use a low power laser as the illuminator, which meets American National Standards Institute requirements for safe use in the eye.1 The detector digitizes the incoming photons for digital imaging. Similar to earlier confocal concepts, two pinholes are used, each placed before the laser and detector. Likewise, the focal points of both are identically calibrated. Optical lenses then focus the laser light to allow Z-axis scanning. However, instead of using the Nipkow disk to scan within the X-Y plane, the laser light reflects off a dichroic mirror, which directs it to an assembly of vertically and horizontally movable scanning mirrors. These motor-driven mirrors scan the laser across the specimen.7,12

Nevertheless, this design does possess some disadvantages. Unlike the tandem scanning design, the laser scanning design can only image one point at a time, decreasing its scan rate significantly. This limitation may decrease its significance in dynamic imaging. In addition, since this system utilizes movable mirrors, optical aberrations become more pronounced as larger angles are required to image a larger field of view.7

Despite these limitations, the laser scanning confocal microscope can produce high quality images. Furthermore, its powerful software can combine the two-dimensional optical sections into a three-dimensional reconstruction.

The confocal microscope concept is a powerful tool that has been used for a long time in ophthalmology, for both

research and clinically applications. Because of it transparency, the cornea is an ideal tissue to study in vivo, using this technique.

Confocal Microscopy Appearance

of Corneal Anatomy

The in vivo microscopic examination of the cornea’s avascular structure is more complex then its transparency would suggest. Therefore, a comprehensive knowledge of this structure is required to truly appreciate confocal microscopy’s power to view corneal anatomy in vivo.

Epithelium

For review, the anterior surface of the cornea is covered with a layer of nonkeratinized, stratified squamous epithelium. This epithelium is comprised generally of three layers, namely, basal, wing and superficial cell layers. As compared to the entire cornea, the epithelium is relatively thin. The average corneal epithelium thickness as measured with a laser scanning confocal microscope (LSCM) was found to be 54 ± 7 μm centrally and 61 ± 5 μm peripherally.17 This difference can be explained by the back surface of the cornea having a smaller radius of curvature than the front surface. This curvature difference also explains why the central cornea is thinner than the periphery. The average central corneal thickness is 545 ± 25 μm and 652 ± 75 μm peripherally.17

The corneal epithelium is a highly metabolically active tissue that continually replenishes itself throughout life (See also Chapter 2, Corneal Physiology). The source of this proliferation emanates from the perilimbal stem cells. It is generally accepted that these stem cells go on to differentiate into basal cells that form the replicating layer that replenishes the epithelium.

Overall, the epithelium is generally comprised of three layers. First, the basal epithelial layer, derived from the perilimbal stem cells, migrate onto the cornea and subsequently differentiate into polygonal wing cells. These wing cells comprise the intermediate cell layers that eventually differentiate into the superficial epithelial cells.

Interestingly, the superficial and basal cell density are in a ratio of 10:1, respectively.17 The mean surface areas of superficial and basal cells as measured by one study are 624 ± 109 μm2 and 66 ± 5 μm2 respectively, with a ratio of 11.0 ± 4.5:1.18 Various studies have shown no correlation with cell density and age. 17,18

Basal Cells

Basal cells measure approximately 10-15 μm in diameter and are generally uniform in size and reflection.18 These basal cells comprise a monolayer, situated at the base of the epithelium. Unfortunately, these basal cells reflect light poorly. For its power TSCM does not image this cell layer well.18 When imaged, the cell borders are visible as highly reflective outlines and highly reflective cell nuclei are visible.15,18 The cell cytoplasm is poorly imaged by the TSCM (Figure 8-4). The LSCM was no better at visualizing basal cell detail than the TSCM.17 Like the TSCM, the LSCM demonstrated morphologically well-defined cells with bright borders.17

The LSCM did show a central basal cell density of 8996

± 1532 cells/mm2 compared to 5699 ± 604 cells/mm2 as measured with TSCM.15,17 Furthermore, the peripheral basal cell density was determined to be 10139 ± 1479 cells/mm2 using the LSCM.17 Thus far, various studies have not demonstrated a statistically significant relationship between gender or age and the basal cell density.15

Figure 8-4: Epithelial basal cells. The highly reflective cell borders with occasional nuclei can be seen in this confocal image. Note that the cell cytoplasm is poorly visible due to low reflectivity.

Wing Cells

Overlying the basal cell layer are two to six layers of polygonal cells called wing cells. These differentiating cells appear uniform in shape and size with dark cytoplasm and bright borders similar to basal cell morphology as seen with confocal microscopy.15,17 Like the basal cells, wing cell details cannot be easily imaged due to poor

reflectivity.15, 17 In general, wing cells tend to be larger than basal cells, but smaller than superficial cells. The average number of cells decrease to 5070 ± 1150 cells/mm2 centrally and 5582 ± 829 cells/mm2 peripherally as measured by the LSCM.17

Superficial Epithelial Cells

The last step in differentiation creates the superficial epithelial cell layer. Generally, this cell layer is approximately one to two cells thick. The cells within this layer appear flat and polygonal on standard light microscopy. With white-light based confocal microscopy, these cells demonstrate light cell boundaries with bright nuclei (Figure 8-5).9,15,18 In addition, these cells tend to vary in size with the largest measuring 50 μm in one study.17 Conversely, one study used a LSCM that imaged epithelial cells with clear cell borders, bright cytoplasm and black nuclei.17 Both techniques showed cells that varied greatly in reflectivity, ranging from dark to bright cells.17 Generally, the bright cells represent metabolic active epithelial cells while the dark cells signify dead or desquamated epithelial

cells.9,15,17,18

Figure 8-5: Epithelial superficial cells. These cells appear flat and polygonal with faint reflective cell boundaries and bright, prominent nuclei. Note that the cytoplasm is poorly reflective.

The average central cornea superficial cell density was measured to be 840 ± 295 cells/mm2 via LSCM and 1213 ± 370 cells/mm2 via TSCM.15,17 Peripherally, LSCM measured 833 ± 223 cells/mm2.

Bowman’s Layer

Bowman’s layer is posterior to the basal cell lamina. It is an acellular layer comprised of randomly dispersed collagen fibrils approximately 12 μm thick. On confocal microscopy, Bowman’s layer appears as an amorphous membrane that is usually difficult to discern.15 This layer usually is identified with the aid of anatomical landmarks. For

example, the subepithelial nerve plexus lies just posterior to the Bowman’s layer. This plexus does image well on confocal microscopy and can be used indirectly to identify Bowman’s layer. This nerves plexus have a beaded appearance on confocal microscopy.15 (For additional details, see “Corneal Innervation” in this chapter).

Stroma

The stroma comprises approximately 90% of corneal thickness. This layer is mostly comprised of keratocytes arranged in parallel lamella and ground substance. However, the stromal structure is not uniform throughout the cornea. For example, the orientation of the collagen fibrils differs in the anterior one-third and posterior twothirds of the corneal stroma and at the limbal regions of the cornea. These fibrils have an oblique arrangement in the anterior one-third of the corneal stroma, while they are parallel in the posterior two-thirds of the stroma and at the limbus they have a circumferential arrangement.19

Just posterior to the Bowman’s layer is the highest concentration of stromal keratoctyes. Mustonen et al15 used the scanning slit confocal microscopy to estimate a density of 1058 ± 217 cells/mm2. Furthermore, they found that these keratocytes progressively decreased in density to approximately 771 ± 135 cells/mm2 in the posterior stroma. Patel and co-workers20 verified this pattern. Additionally, they used the TSCM to calculate keratocyte volume-density in cubic millimeters. The results of this study are shown in

Table 8-1.20

TABLE 8-1: Normal central human keratocyte density with respect to corneal depth. *

* Table adapted from Patel SV et al who used the TSCM to generate this data20

Full-thickness central keratocyte density was negatively correlated with age that decreased 0.45% per year. Interestingly, this finding is similar to the estimated 0.6% per decrease in endothelial cells.21

Unfortunately, stromal keratocyte’s cytoplasm, cell boundaries and collagen substance absorb most of the

Figure 8-6: Anterior stromal keratocytes. The bright reflections in the image represent prominent keratocyte nuclei suspended within a dark, amorphous, non-reflective ground substance. Note, sometimes a small portion of keratocyte cell body can be seen as a reflective outline.

Figure 8-7: Tangential view through the basal epithelium and anterior stroma. The reflective epithelial basal cell outlines can be seen in the upper right. The highly reflective keratocyte cell nuclei with a small portion of the cell body can be seen suspended within a dark, amorphous ground substance in the lower left. Bowman’s layer appears as an amorphous membrane between the stromal keratocytes and epithelial basal cells.

Figure 8-8: Corneal nerve. Numerous keratocytes can be seen with a large, reflective nerve bundle weaving in between the nonreflective collagen fibrils at the top of this image.

In general, the cornea is estimated to possess approximately 2.4 million keratocytes.22 Various studies, using fluorescent antibodies suggest that there may be at least three different types of keratocytes. These keratocytes’ product namely, collagen, is arranged in varying patterns, depending on the stromal depth. These varying arrangements are not likely to be seen in vivo as previously described.

Descemet’s Membrane

Descemet’s membrane represents the basal lamina of the corneal endothelium that thickens throughout life from 3.0– 4.0 μm at birth, to 10.0 – 12.0 μm during adulthood. More specifically, it has two layers, an anterior banded zone that develops in utero and a posterior nonbanded zone that is laid down by corneal endothelium throughout life.23 Unfortunately, the lack of cell nuclei makes Descemet’s membrane indistinguishable on confocal microscopy. The confocal microscopy can only image the surrounding keratocyte nuclei and endothelial cells, which can serve as anatomical landmarks, unless Descemet’s membrane thickens and develops fibrosis.15

Endothelium

The endothelium is a monolayer of hexagonal interdigitated cells. There are approximately 500,000 cells, giving an average cell count of 3055 ± 386 cells/mm2.15 However, since these cells cannot replicate, the absolute cell number decreases with age. Endothelial cell loss tends to be higher during childhood, but stabilizes after age 18.21

Prior cross-sectional studies of adults have demonstrated an annual endothelial cell loss rate of 0.3% to 0.5%.21 However, a recent longitudinal study over 10 years demonstrated a slightly higher rate of cell loss, namely, 0.6% ± 0.5% per year in adults.21 Correlation between age and the rate of cell loss over the prior ten years in this longitudinal study was not statistically significant.21 In other words, the rate of cell loss appears to be constant throughout adulthood. Additional longitudinal studies over a shorter duration demonstrated between 0.3% and 1.0% cell loss per year.21Anterior segment surgery has been implicated in exacerbating the rate of cell loss. For example, three to five years after a penetrating keratoplasty, the measured endothelial rate of cell loss was 7.8%.24 However, this loss may likely represent the donor cells migrating onto the host, thus decreasing its absolute cell density.

Morphologically, the apical surface of these cells faces the anterior chamber and their basal surface produce the posterior banded zone of the Descemet’s membrane. On confocal microscopy, the endothelial cells usually appear as bright cell bodies with dark, hexagonal cell boundaries (Figure 8-9).15 This appearance is similar to that of specular microscopy. Confocal microscopy is a useful clinical tool, in the diagnosis and follow-up of cases with endothelial decompensation as in Fuchs’ corneal endothelial dystrophy.

Figure 8-9: Endothelial cells. Normal endothelial cells characteristically appear as bright cell bodies with dark, hexagonal cell boundaries. Endothelial cell nuclei are not normally visible.

These diseased corneal endothelial cells have a characteristic appearance on confocal microscopy. For review, guttata, the clinical sign for Fuchs’ corneal endothelial dystrophy, likely represents focal collagen accumulation onto Descemet’s posterior surface by abnormal endothelial cells.25 However, this accumulation may also appear with normal aging corneal endothelial

Figure 8-10: Fuchs’ endothelial dystrophy. Multiple hyporeflective areas varying in size surrounded by hyperreflective structures can be seen, which are typical in Fuchs’ endothelial dystrophy. Many of these hyporeflective, round zones contain a central highlight. These dark, hyporeflective areas with occasional central highlights represent cornea guttata.

cells that is unrelated to an underlying disease state. On confocal microscopy, these cells display both polymegathism and pleomorphism. Usually, there are multiple hyporeflective areas varying in size surrounded by hyperreflective structures.25 These irregular bright bodies are typical in Fuchs’ endothelial dystrophy (Figure 8-10).

Many of these hyporeflective, round zones have a central highlight.25 In addition, other hyperreflective areas consistent with fibrous proliferation can also be imaged.25 These dark, hyporeflective areas with occasional central highlights represent guttata.25 Like the stroma, collagen reflects light poorly, and thus, appears dark on confocal microscopy; hence, the reason why guttata appear dark.

Corneal Innervation

The cornea, especially the epithelium, is one of the most densely innervated superficial tissues in the human body. For comparison, the corneal innervation is 20 to 40 times that of tooth pulp and 300 to 600 times that of skin.26 Overall, the central two-thirds of the corneal epithelium is equally, densely innervated that gradually decreases five to six times toward the periphery.26

For review, corneal innervation originates from the ophthalmic portion of the trigeminal nerve and eventually enters the eye along the long ciliary nerve within the perichoroidal space. Branches of this nerve bundle exits just posterior to the limbus and combine into an annular nerve plexus around the limbus. Approximately sixty to

eighty branches radiate in a radial pattern into the substantia propria of the perilimbal conjunctiva and then pierce mostly into the anterior one-third of the corneal stroma.27 These nerve bundles lose their perineurium and myelin sheaths within approximately one millimeter of the limbus. These nerves then branch either dior trichotomously or in a T-like division within the stroma.27

The details of this neural network had been elaborated by one study, using fluorescent antibodies. Most of these stromal nerve bundles were found to combine into a subepithelial plexus (SEP) just posterior to the Bowman’s layer.27 The corneal epithelium is thought to receive its innervation directly from this subepithelial plexus (Figure 8-11). This study found that terminal branches, measuring 10 to 12 μm in diameter, emanated from this plexus, penetrated Bowman’s membrane, weaved among basal cells and combined again to form another nerve plexus, named the basal epithelial plexus (BEP).26,27

Figure 8-11: Subepithelial nerve plexus. The highly reflective lines in this confocal image represent the subepithelial nerve plexus. A recent study concluded that this plexus may lie just posterior to Bowman’s layer. 27 The corneal epithelium is thought to receive its innervation directly from this subepithelial plexus. The majority of nerve fibers in the subepithelial nerve plexus have been described as C-fibers26 that respond to mechanical, chemical, and thermal stimuli.

These corneal nerve bundles are composed of myelinated A δ-fibers and unmyelinated C-fibers.28 Both fiber types, known as nociceptors, can be activated by mechanical, chemical, and thermal stimuli.26,28 The majority of nerve fibers in the SEP of the human cornea have been described as C-fibers.26

Generally, from studies using electron microscopy, the architecture of the BEP has been elucidated. The large nerve bundles within the BEP were found to travel in the nine-to- three clock-hour direction. From these nerve bundles, smaller intermediate bundles bifurcated and traveled at almost right angles in the twelve-to-six clock direction.26

From these intermediate bundles, smaller bundles bifurcate at right angles, traveling again in the nine-to-three direction.26

Confocal laser fluorescence microscopy with threedimensional reconstruction has been able to further elucidate corneal nerve architecture. Morphologically, A δ- fibers (1-5 μm) differs from C-fibers by possessing characteristic bulbous-like thickenings (~5-10 μm) that consistently remain below the basal epithelial layer.26,27 Many of these nerve fibers appear similar to a string of pearls.26,27 These fibers further divide diand trichotomously, resulting in usually five to six fibers that are partly interconnected.27

C-fibers (0.2-2 μm), on the other hand, form short, branching clusters that run mostly perpendicular to the BEP, approaching the superficial epithelial cells.26,29 Furthermore, dichotomous dividing or bulbous-like thickenings associated with A δ-fibers were not found for C-fibers. Directly anterior to the Bowman’s layer, the C- fibers kink and travel within the BEP for only a short distance. Subsequent to traveling 8-10 μm after exiting the BEP, some of these fibers further divide dichotomously. These fibers then branch into fine fibers that weave their way to the superficial epithelial cell layer. They terminate blindly underneath the surface epithelial cell layer. It is estimated that there are approximately 16,000 nerve endings per one mm2 within the superficial epithelial layer.27

General Corneal Characteristics

These confocal microscopes have also detailed the corneal thickness. For example, the average corneal thickness as measured with a LSCM is 545 ± 25 μm centrally and 652 ± 75 μm peripherally.17 Moreover, Patel and co-workers used TSCM to estimate each corneal layer’s thicknesses as shown in Table 8-2.20

Conclusion

The confocal microscope has advanced our understanding of living systems, especially the cornea. This type of microscope has produced impressive photomicrographic images with excellent image resolution and contrast, and with dynamic in vivo scanning capabilities. In addition to research purposes, this microscope has the potential to make clinical diagnoses without performing corneal biopsies. For example, bacterial, fungal and acanthamoeba keratitis have all been detected in vivo without using stains or dyes.1 However, further studies still must be completed to validate this modality as a diagnostic tool. 1

The future role of confocal microscopy may likely become more significant with advances in computer, lighting and camera technology. However, the expense of this system has limited its use to mainly research purposes within the ophthalmologic community. As manufacturing costs decrease and new diagnostic roles are defined, the confocal microscope will become a more common tool in the ophthalmologist’s armamentarium.

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