Ординатура / Офтальмология / Английские материалы / Essentials in Ophthalmology Cornea and External Eye Disease_Reinhard_Larkin_2005

Fig. 13.34. A B.N., 12 years old: confocal microscopy of the epithelium in vicinity of the superficial cells (depth: 5 mm) showing cystic structures with spherical, highly reflective contents similar to the histologic

Fig. 13.35. Histologic findings in Meesmann’s dystrophy (after Naumann: Pathologie des Auges [49]) with intraepithelial cysts containing cellular debris

sections (see Fig. 13.35). B B.N., 12 years old: confocal microscopy of the epithelium at a depth of 30 mm with increased visualization of spherical highly reflective and cystic structures

13.5.3

Epithelium in Contact Lens Wearers

Distinct changes in corneal morphology, pachymetry and structure in contact lens wearers can be demonstrated by in vivo confocal laser scanning microscopy. These findings are best interpreted as resulting from mechanical or metabolic disturbances of the cornea.

All cell layers (superficial, intermediate and basal cells) are present and characterized by bright cell borders and uniformly dark cyto-

plasm. The cell count increases with layer depth due to a decrease in cell diameter. Bowman’s membrane and the subepithelial plexus display border structures between epithelium and stroma (Fig. 13.36A).

Superficial cells are characterized by a dark nucleus, and the cytoplasm is generally darker than in the normal cornea. The polygonal structure is retained, but cell bodies are generally smaller (30 mm in contact lens wearers and up to 50 mm in the normal cornea) (Fig. 13.36B).

Our data (unpublished results) show a significant increase in superficial cell density (p<0.05) both centrally and peripherally.

The intermediate cells do not show any morphological changes by comparison with findings in normal subjects: pale cell borders, invisible nucleus and dark cytoplasm were detected in both the lower and upper wing cells (Fig. 13.36C). A significant reduction in the cell count was noted only in the periphery (p<0.05).

A

C

Basal cell structure is characterized by an inhomogeneous cytoplasm and invisible nucleus; cell diameters are approx. 8–10 mm (Fig. 13.36D). A significant reduction in the cell count was also detected in the peripheral cornea (p<0.05).

Analysis of the pachymetry data revealed reduced corneal thickness in the periphery compared to that in normal volunteers, especially in patients who had worn contact lenses for longer than 10 years. There were no age-related changes in cell count or epithelial thickness, but stromal thickness was reduced.

The type of contact lens (hard vs. soft) has no influence on corneal morphology; duration of contact lens wear was the factor with the great-

B

Fig. 13.37. Micromorphologic changes in the cornea of contact lens wearers: A corneal microdeposits; B signs of polymegathism, pleomorphism and endothelium precipitates; C signs of polymegathism and pleomorphism

est impact. Corneal microdeposits in stroma (Fig. 13.37A) and signs of polymegathism, pleomorphism (Fig. 13.37B, C) and endothelium precipitates (Fig. 13.37B) are the most common findings.

As demonstrated in Fig. 13.38, alterations in Langerhans’ cells also occur due to contact lens wearing.

In light of this,in investigations of the cornea in contact lens wearers, attention must focus on the cell density of each layer and on the thickness of the corneal epithelium, and results must always be compared between the center and periphery.

13.5.4

Epidemic Keratoconjunctivitis

Epidemic keratoconjunctivitis (EKC) is a highly contagious infection caused by type 8, 19, 37 adenoviruses; one of its chief complications is the development of nummular areas of subepithelial corneal opacity which, in exceptional cases, may lead to years of reduced visual acuity and to increased glare sensitivity [30, 69]. Histopathologic examination reveals that the nummular lesions consist of an accumulation of cells from the monocyte-macrophage system, such as lymphocytes, histiocytes and fibroblasts [18].

Viral persistence in the keratocytes is suspected as the cause for the continuing presence of the nummular lesions. The immune response induces focal infiltration of immune cells around the infected keratocytes. The complexes

form the slit-lamp microscopic substrate of the nummular lesions [57] (Fig. 13.39A, B).

Hyperreflective punctate structures can be visualized in the intermediate layer of the epithelium on confocal Rostock laser-scanning microscopy (Fig. 13.40A). These may be lymphocytes, histiocytes and/or fibroblasts [33].

By contrast with physiologic findings, the basal cell layer is hardly distinguishable as such. In addition to a network of hyperreflective dendritic structures (Fig. 13.40B), which becomes clearly less dense with increasing depth (Fig. 13.40C, D), corpuscular changes with dendritic extensions are visualized (Fig. 13.40C, D), some of which appear to be spread out between the nerve fibers (Fig. 13.40E). Considering their location, size and shape, these are most probably the antigen-presenting Langerhans’ cells [59], which are responsible for the induction of cell-mediated delayed-type immune responses. They assume an important role in triggering

Fig. 13.40 a–e. Confocal image of the central cornea in epidemic keratoconjunctivitis; edge length of the image in vivo,250 mm; focal planes moved axially from epithelium to endothelium. a Intermediate epithelial layer with isolated hyperreflective round structures

contact allergies, rejection reactions and viral defense, and in the healthy cornea they are located in the epithelial layers of the conjunctiva, the limbus and peripheral cornea, but not in the central cornea. Migration of the Langerhans’ cells into the central cornea may occur in response to traumatic, chemical or inflammatory stimuli [1, 18].

The changes visible beneath the nerve fiber layer are possibly scatter artifacts in the vicinity of the ruptured Bowman’s membrane [33].

The superficial epithelial layer, stroma and endothelium do not display any abnormalities.

located between the cells; b basal cell layer with hyperreflective dendritic network; c, d transition from basal cell layer to nerve plexus layer with dendritic cell structures, some of which appear to be spread out between the nerve fibers; e Bowman’s membrane

13.5.5

Acanthamoeba Keratitis

Numerous free-living phagotrophic amoebae cause opportunistic infection in humans. Acanthamoeba keratitis has been recognized as a potentially blinding disease, which is often only diagnosed at a late stage. The condition is sometimes confused with other types of infectious keratitis, particularly those of fungal and herpetic origin.

Although not widely available, the confocal microscope can be helpful in establishing the diagnosis of Acanthamoeba keratitis, based on the visualization of pear-shaped cysts approx. 10 mm in length and irregular trophozoites [38, 55] (Figs. 13.41–13.43).

In vivo confocal microscopy permits identification of Acanthamoeba cysts in the cornea [40, 2]. The identity of findings with those from conventional ex vivo microscopy and PCR provides a basis for simple and reliable in vivo diagnosis (Fig. 13.44).

Fig. 13.44 A–C. Confocal microscopy as a non-inva- sive diagnostic method for in vivo identification of Acanthamoeba cysts in the cornea. The identity of findings with conventional ex vivo microscopy pro-

vides a basis for easy and reliable in vivo diagnosis of Acanthamoeba cysts. A Light microscopy in vitro; B confocal microscopy ex vivo; C confocal microscopy in vivo

13.5.6 Corneal Ulcer

Little experience has been gained with confocal microscopy in unspecific corneal ulcers. Leukocyte infiltration may be demonstrated in the ulcer margins in both the epithelial and the

superficial stromal region. Figure 13.45 is a slitlamp photograph and Fig. 13.46 shows confocal microscopy. In vivo confocal microscopy of corneal ulcers provides additional information about corneal healing processes, and permits evaluation of epithelialization and reinnervation at the cellular level.

Fig. 13.46. A Oblique section of central cornea near the ulcer: regular epithelial structure, absence of subepithelial plexus, and distortion of anterior stroma; B at the level of deeper intermediate and basal cells: bright cellular structures, most probably leuko-

cytes; C distortion of basal cell layer and other structures of the subepithelial plexus; D anterior stroma in ulcer area: keratocytes or cell nuclei are not visible, severe destruction of stromal structure

Fig. 13.47. Slit-lamp photograph from a 25-year-old male patient 1 year after laser in-situ keratomileusis (LASIK): uncorrected visual acuity 20/20, normal corneal morphology apart from a small circular stromal scar, representing the border of the former flap zone (arrow)

13.5.7

Refractive Corneal Surgery

The different methods of refractive corneal surgery are designed to reduce ametropia, where present. Depending on the technique used, refractive corneal surgery may result in morphologic changes and sometimes also in irritation and complications in the vicinity of the corneal epithelium or stroma that may lead to subjective disorders [56, 50]. The morphology and mechanism of wound healing processes following refractive corneal surgery are therefore of particular interest in this context [25, 15] (Figs. 13.47–13.49).

In vivo confocal microscopy of the cornea after refractive surgery yields information about the functional status of the keratocytes and the reinnervation of stroma and epithelium [37]. It is possible to define the precise depth location for corneal opacities and to measure changes in corneal thickness [45]. Even years later, the depth of the interface zone following laser insitu keratomileusis (LASIK) can be identified on the basis of the morphologic changes visible there.

Fig. 13.49. Epithelium and keratocytes after LASIK

13.6

Future Developments

13.6.1

Three-Dimensional Confocal Laser-Scanning Microscopy

Many researchers have investigated the cornea with in vivo confocal microscopy [4, 22, 23, 24, 37,72]. This sophisticated tool has been useful in augmenting our understanding of anatomy in the healthy and diseased human cornea. The limitations in magnification due to slight, unavoidable eye movements are obvious and therefore 3-D reconstruction is restricted on practical grounds. The step size is too coarse and magnification is too small. However, 3-D visualization and modeling would improve our understanding of the morphology of corneal architecture, e.g., of epithelial nerve structure.

This was our motivation for developing a fast, non-invasive, high-resolution method for

the detailed, 3-D investigation of the human cornea. The further development of the confocal microscope [79] took the form of a modified confocal laser-scanning ophthalmoscope [66] based on a commercially available instrument (Heidelberg Retina Tomograph II, Heidelberg Engineering GmbH, Germany) [62]. A waterimmersion microscope lens (Achroplan 63¥/ 0.95W/AA 2.00 mm, Carl Zeiss, Germany) with a long working distance and high numerical aperture was used and coupled to the cornea via a PMMA cap by interposing a transparent gel (Vidisic, Mann Pharma, Germany) for in vivo imaging [66]. For 3-D imaging,an internal scanning device moves the focal plane perpendicularly to the x-y plane, in the same way as in optic disk tomography performed with the original HRT II configuration. During image capture the z-movement is stopped and the image plane is exactly perpendicularly to the z-axis. For the investigations presented, an acquisition time of 1 s with a scanning depth of 30 mm was used for all subjects; this is