keratocon

C L I N I C A L

A N D

E X P E R I M E N T A L

OPTOMETRY

INVITED REVIEW

New perspectives on keratoconus as revealed by corneal confocal microscopy Clin Exp Optom 2008;

91 : 1: 3 4 – 55

Nathan Efron* BScOptom PhD DSc Joanna G Hollingsworth† BSc(Hons) PhD

MCOptom * Institute of Health and Biomedical Innovation and School of Optometry, Queensland University of Technology, Technology, Brisbane, Australia † Optometry Giving Sight, Association of Optometrists, London, United Kingdom E-mail: [email protected]

Submitted: 1 May 2007 Revised: 20 June 2007 Accepted for publication: 6 July 2007

DOI:10.1111/j.1444-0938.2007.00195.x Confocal microscopy (CM) of keratoconus is reviewed. In the Manchester Keratoconus Study (MKS), slit scanning CM was used to evaluate 29 keratoconic patients and light microscopy (LM) was performed on two of the keratoconic corneas post-keratoplasty. The findings of the MKS are compared with other CM studies. Consideration of the differences between studies of cell counts is confounded by the use of different experimental controls. A consensus exists among studies with respect to qualitative observations. The epithelium appears more abnormal with increasing severity of keratoconus. In severe disease, the superficial epithelial cells are elongated and spindle shaped, epithelial wing cell nuclei are larger and more irregularly spaced and basal epithelial cells are flattened. Bowman’s layer is disrupted and split in the region of the cone and intermixed with epithelial cells and stromal keratocytes. Stromal haze and hyperreflectivity observed with CM correspond with apical scarring seen with the slitlamp biomicroscope (SLB). Hyper-reflective keratocyte nuclei are thought to indicate the presence of fibroblastic cells. Increased haze detected with CM is found with LM to be due to fibroblastic accumulation and irregular collagen fibres. Dark stromal bands observed with CM correlate with the appearance of Vogt’s striae with SLB. Desçemet’s membrane appears normal with both CM and LM. Some evidence of endothelial cell elongation is observed with CM. The application of CM to ophthalmic practice has facilitated a greater understanding of medical and surgical approaches that are used to treat keratoconus. This review offers new perspectives on keratoconus and provides a framework, against which tissue changes in this visually debilitating condition can be studied in a clinical context in vivo using using CM.

Key words: confocal microscope, cornea, keratoconus, keratocyte

Up to the end of the 20th Century, our clinical view of tissue compromise in living patients with corneal disease was restricted to what could be observed macroscopically and under magnification of up to ×40 using a slitlamp biomicroscope micros cope (SLB). (SLB). In the case of keratoconus, keratoc onus, the SLB serves as an invaluable tool for examining gross tissue Clinical and Experimental Optometry 91.1 January 2008 34

changes such as apical scarring, Vogt’s striae, Fleischer ring (iron deposits around the base of the cone), corneal thinning, protrusion and hydrops. 1 Other techniques such as retinoscopy, 2 corneal topographic analysis, 3 pachometry 3 and optical coherence tomography 3 can assist in the diagnosis and characterisation of this condition.

The relatively recent introduction of the corneal confocal microscope (CM) has dramatically changed the ophthalmic clinical landscape, allowing eye-care practitioners to non-invasively view the living human cornea at magnifications of up to ×700.4 Whereas the SLB facilitates obser vation of the three basic corneal layers— the epithelium, stroma and endothe© 2007 The Authors

Journal compilation © 2007 Optometrists Association Australia

Corneal confocal microscopy of keratoconus Efron and Hollingsworth

Cornea plana

Normal cornea

Keratoconus

Keratoglobus

Figure 1. Normal and abnormal forms of the human cornea, progressing (left to right) from the flattest to the steepest corneal forms

lium—the CM allows the cornea to be observed in vivo at a cellular level. 4 Specifically, it is possible to observe individual cells and cell nuclei in the various layers of the epithelium and endothelium,5,6 cell borders and nuclei of stromal keratocytes, Langerhans cells, 7 the fine epithelial sub-basal nerve plexus 8 and newly-discov newlydiscovered ered features such as stromal ‘microdots’.9 Pathogens such as acanthamoeba 10 and fungi 11 can also be seen in diseased eyes. This technology is allowing researchers and clinicians to embark on a new journey of discovery that th at is re resu sult ltin ingg in a mo more re co comp mple lete te and deeper understanding of the living human cornea in health and disease and may pave the way towards the development of new medical and surgical approaches to the treatment of corneal disease. The human cornea can manifest in a variety of abnormal shapes (Figure 1). This review will focus on research that has employed CM to characterise the clinical histopathology of the keratoconic cornea. Kera Ke rato toco conu nuss is cl clas asssic icaall llyy co con nsi side dere red d as an asymmetrical, progressive, noninflammatory disorder causing an axial corneal ectasia. It is characterised by stromal thinning and corneal steepening, leading to irregular astigmatism and myopia, which cause a marked distortion in vision.1,12 Ultrastructural studies 13–16 have

demonstrated tissue pathology at all levels of the keratoconic cornea. This paper will review recent studies that have used CM to examine patients suffering from keratoconus, to construct a comprehensive model of the ‘clinical histopathology’ of the living keratoconic cornea. Consideration will also be given to the use of CM to develop a greater understanding of the keratoconic cornea with concurrent disease and medical and surgical interventions for keratoconus. THE MANCHESTER KERATOCO KERATOCONUS NUS STUDY

The Manchester Keratoconus Study (MKS),17–20 which will form the cornerstone of this review, is a recently completed examination of 29 keratoconic patients (mean age 31 ± 10 years; range 16 to 49 years), who presented consecutively to the outpatient clinic of the Royal Eye Hospital in Manchester, UK. Some patients were new to the clinic (referred for a definitive diagnosis) and some were existing patients. Both eyes of all patients were examined, however however,, due to three patients having unilateral disease and a further four patients having previously undergone penetrating keratoplasty, data were obtained from 51 eyes. The mean age at diagnosis, as reported by the patients, was 21 ± 8 years (range 4

© 2007 The Authors Journal compilation © 2007 Optometrists Association Australia

to 42 years). Male patients made up 76 per cent of the study group. A family history of keratoconus was present in 17 per cent of patients. The majority of patients (62 per cent) were Caucasian, 24 per cent were of Asian descent and 14 per cent were AfroCaribbean. Ninety per cent of patients had bilater bilateral al keratoc keratoconus onus and 52 per cent reported report ed that they suffered suffered from some form of atopy atopy,, such as asthma, asthma, eczema, eczema, hay fever, general allergies or a combination of these. One patient also suffered from retinitis pigmentosa. There were no other associated systemic conditions. A large number of patients reported a history of eye rubbing (66 per cent). Fifty per cent of eyes were currently fitted with rigid contact lenses, the majority of which showed some level of apical touch (64 per cent). Thirty-three per cent of eyes were corrected with spectacles. One patient was fitted bilaterally with scleral contact lenses. Three patients were awaiting surgery and were no longer using any form of visual correction.

Clinical investigative techniques SLITLAMP BIOMICROSC BIOMICROSCOPY OPY

All eyes were examined with a SLB and 27 per cent of eyes displayed corneal staining, 37 per cent of eyes demonstrated corneal scarring and 37 per cent of eyes had Vogt’ss striae. Vogt’ Clinical and Experimental Optometry 91.1 January 2008 35


CORNEAL TOPOGRAPHY

Corneal topography was attempted on all 51 eyes using the EyeSys 2000 Corneal Analysis System (EyeSys Technologies, Houston, Texas, USA). This enabled disease severity to be classified by corneal curvature, using the same system as used in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) study.21 According to this system, disease severity is classified with respect to the curvature of the steepest corneal meridian as follows: mild, less than 45 D; moderate, 45 to 52 D; and severe, more than 52 D. Corneal topographical maps were produced from 46 eyes. In the remaining five eyes, the cornea was too distorted or scarred to obtain a reading of corneal cur vature; these were classified as severe keratoconus. The average steep keratometric reading was 52.1 ± 7.9 D (range 42.5 to 81.0 D). The majority of patients had either moderate or severe keratoconus; specifically, 12 per cent of eyes exhibited mild keratoconus, 39 per cent of eyes had moderate keratoconus and 49 per cent of eyes were classified as having severe keratoconus. CONFOCAL MICROSCOPY

All keratoconic patients and control sub jects were examined using an in vivo slit scanning real time CM (Tomey Confoscan P4, Erlangen, Germany) fitted with an Achroplan 40X/0.75NA immersion objective. One drop of local anaesthetic (Benoxinate Hydrochloride 0.4%, Chau vin Pharmaceuticals, Romford, UK) was instilled into the lower fornix of the eye.22 A drop of polymer gel (Viscotears, CIBA Vision, Duluth, Georgia, USA) was applied to the microscope probe prior to the examination to optically couple the microscope objective lens to the cornea. CM was performed on the central cornea of all eyes. Images obtained of the corneal layers of the 51 keratoconic eyes during CM examinations were stored on S-VHS videotape. Each examination was evaluated frame by frame by a single examiner. Images were saved of the epithelium, Bowman’s layer, the anterior and posterior stroma, Clinical and Experimental Optometry 91.1 January 2008 36

Desçemet’s membrane (where visible) and the endothelium. In the course of scanning through the cornea along the central anterior-posterior axis, image frames of the anterior stroma were taken to be those immediately posterior to Bowman’s layer and image frames of the posterior stroma were taken to be those immediately anterior to the endothelium. In addition, images showing any abnormality were saved regardless of their position within the cornea. For quantitative analysis, only the right eye of each study participant was examined; however, in cases where it was not possible to use the right eye (such as in unilateral keratoconus or if the right eye had previously undergone penetrating keratoplasty), the left eye was used. Thus, quantitative data were obtained from 29 eyes. Quantitative analysis was conducted using dedicated software that came with the CM (Confocommander 2.7.1, Tomey, Erlangen, Germany). At least three frames were analysed for each corneal layer and an average was taken. When examining images of the endothelium a minimum of 50 cells was evaluated. During the course of the study, it became apparent that the images obtained of the keratoconic stroma were degraded to varying degrees. Many images were hazy and often had a hyper-reflective appearance. Keratocyte nuclei were poorly differentiated in hazy images, rendering analysis difficult. To quantify the levels of haze and hyper-reflectivity, a grading scale of this phenomenon was constructed from representative images (Figure 2). The meaning of each level of grading is explained in Table 1. The level of haze in each stromal image analysed in this work was graded using this tool. HISTOLOGICAL STUDIES

Two of the patients examined in this study (referred to as Patient A and Patient B) went on to have penetrating keratoplasty and the excised corneal buttons were subsequently available for examination using light microscopy (LM). Following surgical removal, the corneal buttons were placed in 10 per cent neutral buffered formalin (pH 7.6). The buttons were cut into two

sections along the vertical meridian, dehydrated through graded alcohols (70, 90 and 100 per cent), de-lipidised in xylene and impregnated with paraffin wax at 56°C. Patient A had unilateral keratoconus. Limited cross-sections of the central cornea of this patient were available and were stained with haemotoxylin and eosin. Serial step sections were prepared from the excised cornea of Patient B and were stained in an alternating manner with haemotoxylin and eosin, Periodic acid-Schiff and Masson’s trichrome. The histological appearance of the corneas of these two patients is considered below in the relevant sections relating to each corneal substructure. THE CORNEA IN KERATOCONUS

Superficial epithelial cells CM images of the superficial epithelial cell layer were obtained in 20 per cent of eyes in the MKS.17–20 A normal appearance5,6 was noted in eight per cent of eyes, all of which were classified as having moderate keratoconus. Irregular superficial cells with an elongated or spindle-like shape were found in 12 per cent of eyes. These patients were classified as having severe keratoconus (Figure 3). The CM appearance of elongated epithelial cells has been observed by others. Somodi and colleagues23 reported seeing obviously elongated superficial epithelial cells arranged in a whorl-like fashion. Wygledowska-Promienska and associates 24 noted that desquamating epithelial cells were elongated and arranged around the apex of the cornea. Uçakhan and colleagues25 observed the epithelial cells to be elongated in 18 per cent of keratoconic eyes (all with severe keratoconus). Desquamating, elongated superficial epithelial cells were observed in one patient. Weed and associates 26 noted that desquamating superficial epithelial cells with bright cell boundaries were easily visible in keratoconic eyes. Uçakhan and colleagues 25 found that the density of superficial epithelial cells in keratoconic eyes (942 ± 137 cells/mm2) © 2007 The Authors



Grade 0

Grade 1

Grade 2

Grade 3

Grade 4

Figure 2. Grading scale for quantifying the level of haze and hyper-reflectivity in CM images of the corneal stroma. The various grades are defined in Table 1.

Grade

Severity

Description

0

Normal

Keratocyte nuclei clearly defined. Accurate analysis possible in all cases.

1

Trace

Keratocyte nuclei visible; some background haze. Accurate analysis possible in most cases.

2

Mild

Some keratocyte nuclei visible; many keratocytes partially obscured by haze. Accurate analysis possible in some cases.

3

Moderate

Keratocytes almost completely obscured by haze. Accurate analysis possible in few cases.

4

Severe

Keratocytes completely obscured by haze; extreme levels of hyper-reflectivity. Accurate analysis not possible.

Table 1. Explanation of grades of level of haze and hyper-reflectivity in the corneal stroma depicted in Figure 2

was not significantly different from that of age and gender-matched controls (1,087 ± 971 cells/mm2). This contrasts with the specular microscopy results of Tsubota and associates, 27 who found the mean size of superficial epithelial cells to be increased in keratoconus. Mocan and Irkec28 proposed that the instillation of fluorescein prior to CM can enhance the imaging characteristics of this technique. They observed increased intracytoplasmic and nuclear staining of the superficial epithelium that was more readily visible after instillation of fluorescein. They suggested that this increased intracytoplasmic and nuclear staining in the superficial corneal epithelium of patients with keratoconus might be indicative of increased epithelial turnover.

Wing cells The wing cell layer of the epithelium appeared normal 5,6 in only eight per cent of eyes, all of which had moderate keratoconus. In patients with severe keratoconus, the wing cell layer displayed large, irregularly spaced nuclei (16 eyes of 12 patients) (Figure 4). No images of the wing cell layer were obtained from the remaining patients. The mean diameter of the wing cell nuclei in the keratoconic patients (9.2 ± 1.0 µm) was significantly greater (p < 0.0001) than that of the normal population (6.4 ± 0.8 µm). Basal epithelial cells CM images of the epithelial basal cell layer revealed considerable inter- and intra-


patient variability. A normal appearance of visible cell borders with a regular arrangement of cells5,6 was found in eight per cent of eyes, all of which were classified as having either mild or moderate keratoconus. The most common finding was a hazy appearance (22 eyes of 16 patients). In 12 per cent of eyes, the basal cell layer had an irregular appearance, with large cells and faint cell borders (Figure 5). These images showed some similarities to the images obtained of the wing cell layer but were differentiated from wing cell images by virtue of their location adjacent to Bowman’s layer. Most of these eyes were classified as having severe keratoconus. The average basal epithelial cell diameter in keratoconic patients (11.4 ± 1.2 µm) was significantly greater (p < 0.05) than that in control subjects (10.4 ± 0.9 µm). This is consistent with the finding of Weed and associates, 26 who found that that basal cell density in moderate keratoconus (4,592 ± 414 cells/mm2) and severe keratoconus (4,530 ± 596 cells/mm2) was lower than that of age- and sex-matched controls (4,912 ± 434 cells/mm2). The opposite was found by Uçakhan and colleagues,25 who reported that basal cell density in keratoconic eyes (11,273 ± 5,009 cells/mm2) was greater than that of age- and sex-matched controls (5,941 ± 1,853 cells/mm2). Consistent with this, Uçakhan and colleagues 25 reported basal cell area to be lower (p < 0.05) in keratoconic eyes (115 ± 49 µm2) compared with non-keratoconic controls (179 ± 97 µm2). The epithelial basal cell densities reported by Uçakhan and colleagues 25 Clinical and Experimental Optometry 91.1 January 2008 37


were significantly higher than those reported by Weed and associates 26 in both keratoconic patients (unpaired t-test: moderate keratoconus t = 3.5, p < 0.001, severe keratoconus t = 4.5, p < 0.0001) and non-keratoconic control subjects (t = 2.1, p = 0.038). The reason for this is unclear but may be related to the type of morphometric analysis used. Perhaps also the authors used different criteria for locating and imaging the basal cell layer; for example, imaging at a slightly more anterior plane in the basal epithelium will result in a slightly lower cell density because cells begin to spread laterally when they progress anteriorly, as part of the normal growth patterns of the epithelium. Notwithstanding these differences, both studies appear to be internally consistent, with keratoconus and control eyes imaged and measured using the same techniques. Thus, the differences between keratoconus and control data within each study must be considered to be valid findings. Brightly reflective deposits were observed by Uçakhan and colleagues 25 within the basal epithelial cells of 44 per cent of keratoconic eyes. This material was thought to represent haemosiderin accumulation corresponding to the Fleischer ring in those eyes. Rokita-Wala and associates29 observed similar changes using CM and suggested that the brightly reflective material represents clusters of iron deposits, based on similar appearances in patients with corneal scars and those who have been subjected to radial keratotomy and photorefractive keratectomy. In the MKS,17–20 LM of the corneas of Patients A and B revealed apparent variations in epithelial thickness across the cornea in keratoconus. The epithelium appeared to be thinner in the central region and thicker towards the inferior cornea (Figure 6). Similar pan-corneal variations in epithelial thickness in keratoconus have been observed using in vitro techniques of light microscopy 30 and transmission31 and scanning 32 electron microscopy. Chi, Katzin and Teng 33 suggested that the primary lesion in the keratoconic eye is located in the basal epithelial cells. They Clinical and Experimental Optometry 91.1 January 2008 38

‘Sp indle -like ’ ep ithe lial c ells

No rmal e pit helia l ce lls

Ir re gu lar & br igh t ep ith elial cells

Figure 3. Top row: CM images of the superficial corneal epithelium A. Elongated spindle-like superficial epithelial cells of Patient B B. Normal superficial epithelium C. Irregular elongated superficial cells (left field) and apparently normal superficial epithelial cells (right field) of Patient B D. Light microscopic image of the apex of the cone of Patient B, showing regions of elongated and irregular epithelial cells that correspond to the CM images ( ¥ 40 objective)

Keratoconus

Normal cornea

Figure 4 A. CM image of large, irregularly spaced nuclei in the wing cell layer of the epithelium in keratoconus B. CM image of normal wing cell layer

observed degenerative changes early in the disease. In the later stages of keratoconus, they noted that basal cells disappear and the epithelium is represented by one

or two layers of flattened superficial cells lying on an abnormal basement membrane, Bowman’s layer or directly on the anterior stroma.33 The LM and CM obser© 2007 The Authors



vations in the MKS 17–20 confirm these histological observations. Tsubota and associates 27 were the first to report the appearance of elongated superficial cells in vivo using the specular microscope. They found elongated superficial epithelial cells, which became sharp and spindle-like in severe cases of keratoconus. Spindle-shaped cells are characteristic of the wound-healing response and its associated cellular migration34 and the presence of such cells has also been noted in patients who have undergone penetrating keratoplasty or epikeratophakia. 35 All of the corneas displaying elongated superficial cells in the MKS 17–20 were classified as severe keratoconus.

Sub-basal nerve plexus Patel and McGhee36 used laser scanning CM to produce two-dimensional reconstructions of the corneal sub-basal nerve plexus in four eyes of four patients with keratoconus. This was achieved by having patients fixate on targets arranged in a grid to enable imaging of the cornea in a wide range of positions. A mean of 402 ± 57 images was obtained for each cornea, to create confluent montages (Figure 7). The mean dimensions of the corneal areas mapped were 6.6 ± 0.7 mm horizontally and 5.9 ± 0.7 mm vertically. Thus, these authors were essentially able to elucidate the overall distribution of subbasal nerves in the living central to midperipheral human cornea in keratoconus. All corneas exhibited abnormal subbasal nerve architecture compared with patterns previously observed in normal corneas.8 At the apex of the cone, there was a tortuous network of nerve fibre bundles, many of which formed closed loops. At the topographic base of the cone, nerve fibre bundles appeared to follow the contour of the base, with many of the bundles running concentrically in this region (Figure 8). Central sub-basal nerve density was significantly lower (p < 0.01) in keratoconic corneas (10,478 ± 2,188 µm/ mm2) compared with normal corneas (21,668 ± 1,411 µm/mm2). Uçakhan and colleagues25 reported that nerve fibres in the sub-basal layer had a thickened, prominent appearance in 14

Keratoconus

Normal cornea

Figure 5 A. CM image of large cells with faint borders and general haze, in the basal cell layer of the epithelium in keratoconus B. CM image of normal basal cell layer

Inferior cornea

Cone apex (scarred)

Thickened epithelium (10 layers)

Superior cornea

Thinned epithelium (3 layers)

Normal epithelium (5-6 layers)

Figure 6. Whole mount LM section of the cornea of Patient B (top), showing regional variations in epithelial thickness (bottom)

keratoconic eyes (29 per cent of their sample). Nine of these eyes had severe keratoconus, four had moderate keratoconus and one had mild keratoconus. The mean sub-basal nerve fibre thickness was 4.1 ± 0.7 µm (range 3.1 to 5.3 µm) in keratoconic eyes and 3.7 ± 0.5 µm (range 3.1 to 4.6 µm) in control eyes. In 31 per cent


of keratoconic eyes, sub-basal nerve fibres showed structural changes, such as excessive branching and curling.

Bowman’s layer Bowman’s layer appeared normal 5,6 in 22 per cent of eyes when viewed with CM, that is, as an amorphous, acellular layer. Clinical and Experimental Optometry 91.1 January 2008 39


B

A

400 µm

Figure 7 A. Schematic showing the architecture of the normal human sub-basal nerve plexus. B. Wide-field CM montage consisting of 428 images, depicting the architecture of the sub-basal nerve plexus in a patient with moderate keratoconus. Reproduced with permission from Patel and McGhee.36

A

B

Anterior tangential power (D) C

D

60.00 57.00 54.00 51.00 48.00 45.00 42.00 39.00 36.00 33.00 30.00

2 cm

Figure 8. Electronic tracings of nerve fibre bundles provide schematics devoid of background data in four keratoconic patients, labelled A, B, C and D. These tracings are superimposed, to scale, onto the corresponding anterior tangential corneal topographical maps of these patients. Reproduced with permission from Patel and McGhee.36 Clinical and Experimental Optometry 91.1 January 2008 40

None of the keratoconic corneas in this group with a normal appearance of Bowman’s layer displayed corneal scarring. All eyes were classified as having moderate or severe disease. Bowman’s layer had an abnormal appearance in the remaining 57 per cent of eyes. The type of abnormality varied between patients. In many cases, both epithelial nuclei and keratocyte nuclei from the anterior stroma appeared to be in the same plane as Bowman’s layer. In some images of Bowman’s layer, nerve fibres appeared to run in and out of the plane of the field of view. An increased level of haze was apparent in many images, which corresponded to increased haze in the anterior stroma. The images of Bowman’s layer from three patients (three eyes) contained hyper-reflective patches. Some images also displayed what appeared to be hyper-reflective nuclei of the keratocytes (seven eyes). These hyper-reflective changes were seen only in patients with apical scarring. In agreement with the findings of the MKS,17–20 Somodi and colleagues23 and Wygledowska-Promienska and associates 24 noted highly reflective changes near Bowman’s layer. Somodi and colleagues 23 also observed fold-like structures. These may have been artefacts induced by pressure of the cone tip of the CM against the corneal surface. Such artefacts have been reported to occur with the use of CMs that require corneal contact 37–39 and the instrument used by Somodi and colleagues 23 required corneal contact. Another explanation for the appearance of fold-like structures is that these may have been so-called ‘Kstructures’, which are features that have been reported to appear in the region of Bowman’s layer in the normal cornea. 40 Contrary to the above observations, Uçakhan and colleagues25 and Weed and associates26 were unable to detect any abnormalities in Bowman’s layer in their keratoconic patients. In the MKS, 17–20 histological examination of the cornea of Patient B revealed Bowman’s layer to progress from a normal single layer to an abnormal bilayer, as it approached the apical region and to become split and fragmented in the © 2007 The Authors



immediate proximity of the apical scar (Figure 9A). The bilayer appearance is evident in the corresponding CM image (Figure 9B), in which keratocyte nuclei can be observed to the right of the field and a generally amorphous field is observed on the left, with the odd faint keratocyte nucleus and a single nerve fibre traversing the frame. The normal CM appearance of Bowman’s layer is shown in Figure 9C. With the CM, Bowman’s layer appeared as a hyper-reflective field (Figure 9D) in the heavily scarred region of the cone. Many research groups13,14,16,33,41 have reported the presence of breaks and discontinuities in Bowman’s layer in keratoconus using LM and both transmission and scanning electron microscopy. Sawaguchi and colleagues16 used scanning electron microscopy to examine the keratoconic cornea and found breaks in Bowman’s layer and irregular thinning. These findings are consistent with the appearance of this layer with the CM in the majority of the keratoconic eyes in the MKS.17–20 LM of the cornea of Patient B confirmed that the irregular appearance of Bowman’s layer using CM was consistent with fragmentation and breaks in Bowman’s layer. The ruptured areas of Bowman’s layer have been reported to be filled with either epithelium or proliferated collagenous tissue that is derived from the anterior stroma.13,14,16,33,41 Chi, Katzin and Teng 33 documented keratoblasts and newly formed connective tissue in areas where Bowman’s layer had been destroyed. In the MKS,17–20 LM examination of the cornea confirmed that hyper-reflective nuclei seen with the CM corresponded to fibroblastic cells. Histological examination of the cornea of Patient B demonstrated considerable disruption to Bowman’s layer in the region of the apical scar, which consisted of abnormal collagenous material and fibroblasts. These findings are in agreement with the observations of Chi, Katzin and Teng. 33 These authors described the presence of keratoblasts, however, at the time that their study was conducted, it was not known that keratocytes were able to be activated into a fibro-

A

Scar tissue

B

C

‘Split field’

D

Normal

Hyper-reflectivity

Figure 9. LM image of the apical region of the cornea of Patient B ( ¥ 10 objective). The dotted circle indicates the scarred region of the cone. The arrows indicate the path of Bowman’s layer, which appears as a single layer at the extreme right of the field and then splits into a bilayer. The bilayer separates and then rejoins towards the left of the field to form a bilayer again. The CM images (bottom row) indicate various appearances of Bowman’s layer: B. ‘Split field’ in Patient B C. Normal appearance in a control subject D. Hyper-reflectivity

blastic status. This was demonstrated many years later.42

Stroma Stromal images of the central cornea obtained by CM showed varying amounts of haze and hyper-reflectivity. Extreme levels of haze were present in 44 per cent of eyes. When visible in these corneas, the keratocyte nuclei often displayed an irregular, hyper-reflective appearance. Severe haze was found to correspond with apical scarring on SLB evaluation in 35 per cent of eyes. The four eyes in which apical scarring was not apparent when viewed with the SLB displayed less severe levels of haze on CM. The remaining eyes showed only mild degrees of haze. In these patients, keratocyte nuclei were easily distinguished and had an appearance similar to that seen in the normal eye. 5,6


The level of haze was quantified using the grading scale (Figure 2). The presence of scarring on SLB examination was significantly related to the level of haze seen on CM. This was true for both the anterior (F = 7.6, p < 0.05) and posterior stroma (F = 5.193, p < 0.05). In some cases, the location of scarring observed with the SLB corresponded to the region of apical touch of the rigid contact lens that the patient was wearing. Figure 10 illustrates the co-location of contact lens apical touch and apical scarring in a keratoconic patient (not from the MKS). Regression analysis was employed to investigate the effects of haze on the apparent keratocyte density (KD). Increasing levels of haze were associated with a reduction in the apparent KD. This relationship was of a much higher significance for the anterior (r 2 = 0.52, F = 24.9, p < 0.0001) than the posterior stroma Clinical and Experimental Optometry 91.1 January 2008 41


(r2 = 0.16, F = 4.6, p = 0.04). These findings support the validity of the keratoconic haze grading scale. Surprisingly, the degree of stromal haze was not shown to bear any relationship to disease severity as classified by corneal curvature. Haze in the corneal stroma of keratoconic eyes, especially the anterior stroma, has also been noted by others, in agreement with the MKS. 17–20 Uçakhan and colleagues25 reported increased background illumination and reflectivity, and irregular arrangement of stromal keratocyte nuclei in the anterior stroma of 29 per cent of eyes. They suggested that this appearance was consistent with varying degrees of haze and stromal scarring observed using SLB. Wygledowska-Promienska and associates24 noted an apparent disarrangement of collagen fibres reflected by bright background illumination in the anterior region of the stroma beneath Bowman’s layer. Somodi and colleagues 23 also observed increased reflectivity in the anterior stroma. In the posterior stroma, keratocytes had extremely long almost parallel processes, however, in scarred stroma, the keratocytes were spindle-shaped and arranged irregularly.

Keratocyte density An assessment of stromal KD in keratoconus is confounded by two key factors. First, patients with keratoconus are typically fitted with rigid contact lenses to neutralise corneal distortion and afford satisfactory vision. The more severe the condition, the more likely it is that rigid lenses are being worn. With the exception of one research group,43 the general consensus in the literature is that, in normal subjects, contact lens wear causes an apparent reduction in KD.44–48 This is thought to occur as a result of the physical impact of lenses on the corneal epithelium, which releases inflammatory mediators that cause keratocyte apoptosis.49 Thus, there is a need to determine whether the reduction in KD associated with keratoconus is due to the effects of lens wear or the direct pathological effects of keratoconus or possibly both. Second, as discussed above, the corneal stroma in keratoconus is often hazy and it Clinical and Experimental Optometry 91.1 January 2008 42

Figure 10 A. Central bearing of a rigid lens fitted to a patient with keratoconus, reve aled with the aid of fluorescein B. Same eye as shown in (A) with lens removed. The apical scarring visible within the pupil corresponds to the region of contact lens bearing. This patient is not from the MKS. (Photographs courtesy Ruth Cornish)

is difficult to see keratocytes in the presence of significant haze, leading to a potential under-estimation of KD in such cases. The four studies 19,25,26,44 that have addressed the question of KD in keratoconus adopted different approaches in attempting to account for these confounding influences. Table 2 provides a summary of estimates of KD published in these works.19,25,26,44 In reviewing the data in this table, it should be noted that the absolute cell densities reported by Erie and associates 44 can not be directly compared with the other data displayed in the table because Erie and associates 44 expressed cell density as a volumetric measure (cells/mm 3), whereas all other data are expressed as a function of cell area (cells/mm2). It is not possible to con vert between the two units because the depth of the CM sections used to calculate field volume was not stated by Erie and associates.44 Most of the keratoconic patients in the MKS17–20 were wearing rigid contact lenses, so a control experiment 50 was conducted to determine the effects of rigid lens wear on KD in non-keratoconic subjects. Slit scanning CM (Tomey Confoscan P4) was used to evaluate KD in 22 subjects who had been wearing rigid lenses on a longterm, daily wear basis. These data were

compared to those of 22 non-lens-wearing control subjects. Subjects with a pre vious history of polymethyl methacrylate (PMMA) lens wear showed a reduction (p < 0.0001) in anterior keratocyte density (AKD) and an increased level of haze in the anterior stroma. When the data of all 22 subjects were combined, AKD and posterior keratocyte density (PKD) appeared unaffected by rigid lens wear (p = 0.10 and 0.34, respectively). Therefore, nonlens-wearing non-keratoconic subjects were used as experimental controls in the MKS. In view of the subsequent study by Kallinikos and Efron, 49 which demonstrated a reduction in KD at all corneal depths in patients wearing rigid contact lenses (with no prior PMMA lens wear), the control used in the MKS may have been inappropriate. To remove the confounding effects of haze-induced image degradation on KD in the MKS,17–20 any images classified as having haze of grade 2 or above were removed from the analysis. The mean anterior and posterior keratocyte densities were found to be significantly lower in the keratoconic than control eyes, specifically, the AKD and PKD were 19 and 10 per cent lower than in controls, respectively. Overall, atopy, a history of eye rubbing and the presence of corneal staining were related to AKD (r2 = 0.78, F = 9.4, © 2007 The Authors



Author Erie and colleagues

Year 44

Hollingsworth, Efron and Tullo19 Uçakhan and colleagues 25 Weed and associates 26

2002 2005 2006 2007

Anterior stroma Keratoconus Control b

24,564 ± 8,750 32,724 ± 7,105c 909 ± 91d 879 ± 371c 883 ± 111b,e 952 ± 122b,f 883 ± 111b,e 952 ± 122b,f

Posterior stroma Keratoconus Control

p-value b

35,630 ± 3,858 31,168 ± 6,818c 1,119 ± 80c 1,082 ± 195c 609 ± 66b 609 ± 66b 761 ± 118c 761 ± 118c

p < 0.001 NSg p < 0.001 p < 0.05 p < 0.001 p < 0.001 p < 0.001 p < 0.001

b

11,118 ± 3,454 15,219 ± 5,572c 528 ± 50d 547 ± 95c 550 ± 54b,e 599 ± 97b,f 550 ± 54b,e 599 ± 97b,f

p-value b

18,704 ± 4,313 18,129 ± 3,515c 584 ± 77c 703 ± 109c 470 ± 63b 470 ± 63b 504 ± 80c 504 ± 80c

p < 0.001 NSg p < 0.004 p < 0.05 p < 0.001 p < 0.001 NSg p < 0.001

a

Units of density are cells/mm 3 for Erie and colleagues 44 and cells/mm2 for all other authors Only lens wearers c Only non-lens wearers d Mixture of lens wearers and non-lens wearers e Moderate keratoconus f Advanced keratoconus g Not significant (p > 0.05) b

Table 2. Keratocyte densitiesa in patients with keratoconus reported by various authors

P = 0.005) but not PKD. Post hoc analysis revealed that atopy (p = 0.009), a history of eye rubbing (p = 0.006) and corneal staining (p = 0.035) were each significantly associated with AKD. Contact lens wear, race and the presence of a scar were unrelated to AKD or PKD. Erie and associates44 measured KD in four groups: lens-wearing and nonlens-wearing keratoconic patients and lens-wearing and non-lens-wearing nonkeratoconic control subjects. Among those who did not wear contact lenses, no difference in AKD and PKD was found between keratoconic patients and nonkeratoconic control subjects. Among contact lens wearers, AKD and PKD were found 31 and 41 per cent lower in keratoconic patients compared with nonkeratoconic control subjects, respectively. These authors concluded that KD is normal in keratoconic patients but keratocyte loss is somehow exacerbated by lens wear. As Erie and associates 44 excluded patients with severe keratoconus from their study, interpretation of their results must be confined to changes that occur in mild to moderate keratoconus. In the study of Uçakhan and colleagues,25 only non-lens wearing keratoconic and control subjects were

examined. These authors found AKD and PKD to be 19 and 22 per cent lower than in controls, respectively. Although this finding contradicts that of Erie and associates,38 who found no difference in AKD or PKD between non-lens-wearing keratoconic and control subjects, it should be noted that the keratoconic patients in the experiment of Uçakhan and colleagues 25 were not confined to those with mild to moderate disease. In fact, 54 per cent of their experimental group were classified as having severe keratoconus. Uçakhan and colleagues25 did not explain how their patients with severe keratoconus managed to see, given that they apparently were not corrected with rigid contact lenses or any other form of contact lenses. Considered together, the findings of Erie and associates 38 and Uçakhan and colleagues25 suggest that keratocyte loss in keratoconus may be related to disease severity. Weed and associates 26 assessed patients with moderate and advanced keratoconus who wore contact lenses and found significantly higher KD compared with nonlens-wearing control subjects. Specifically, AKD was 14 per cent higher in patients displaying moderate keratoconus, and AKD and PKD were 20 and 16 per cent


higher in patients with advanced keratoconus. Compared with lens-wearing control subjects, AKD and PKD were 31 and 15 per cent higher in patients displaying moderate keratoconus, and 36 and 22 per cent higher in patients with advanced keratoconus. The findings of Weed and associates26 of higher KD in keratoconic patients directly contradict those of the MKS17–20 of a lower KD in keratoconic patients. In view of the different approaches outlined above in determining KD in keratoconic patients, it is difficult to reconcile the results of these works. Three of the four papers that addressed this issue 19,25,44 indicate a lower KD in keratoconus but the extent to which these changes reflect the effects of lens wear versus the underlying pathological changes in keratoconus remains unclear. One possibility, suggested by Kallinikos and Efron 49 is that changes in KD may be a function of lateral cell migration and redistribution as well as, or instead of, cellular apoptosis. A combination of these phenomena could explain both increases and decreases in KD. The original idea—conceived half a century ago by Chi, Katzin and Teng 13,33— that the earliest ultrastructural changes in Clinical and Experimental Optometry 91.1 January 2008 43


Epithelium Stroma

Receptor for Interleukin-1 Keratocyte

Endothelium

Normal cornea

Epithelial trauma

A

Interleukin-1 B

C

D

Keratoconus

Figure 11. Theory of keratocyte apoptosis in keratoconus. Left: The normal cornea. Keratocytes have receptors for Interleukin-1. Right: The keratoconic cornea. A. Keratocytes have four times as many receptors for Interleukin-1 as a normal cornea B. Epithelial trauma causes a release of Interleukin-1, which floods the cornea C. Most of the Interleukin-1 has left the cornea, but some remains bound to receptors D. Interleukin-1 bound to the receptors induces keratocyte dysgenesis and apoptosis

keratoconus occur at the epithelial basement membrane, has been extended recently by Wilson and colleagues, 45 who suggest that epithelial damage causes a reduction in AKD through apoptosis. This is thought to be triggered by cytokines (including interleukin 1 and Fas-ligand) released from the damaged epithelial cells.51 An increased number of anterior keratocytes exhibit signs of apoptosis in the keratoconic cornea when compared to normal corneas and corneas suffering from other diseases. 52 It has also been shown that the keratocytes within the keratoconic cornea have four times as many receptors for interleukin 1, potentially sensitising them to this cytokine. 53 CM reports of reduced AKD in keratoconus19,25,44 and the LM observations from the MKS17–20 are consistent with the notion that keratocyte apoptosis induced by epithelial damage is one of the mechanisms responsible for the reduction in AKD in keratoconus (Figure 11). The clinical evidence of this is the presence of significant corneal fluorescein staining in keratoconic patients as observed in the MKS.17–20 Clinical and Experimental Optometry 91.1 January 2008 44

Confocal versus light microscopy In the MKS,17–20 hyper-reflective keratocyte nuclei and stromal haze were apparent when examining CM images of the cornea of Patient B (Figure 12). Evaluation of the serial step sections prepared for LM revealed the presence of disorganised tissue, confirming the SLB appearance of apical scarring in this patient. The scarred region measured approximately 220 µm at its widest point. Accurate measurement of hyper-reflective regions in CM images was not possible as there was no defined border, however, the size of the regions of hyper-reflectivity observed with the CM was roughly consistent with measurements of the scar taken from the histological samples. Examination of tissue sections from the cornea of Patient B at higher magnification revealed a dense accumulation of fibroblasts in the region of the scar. Nuclei were rounded and more irregular in shape compared to the elongated, flattened appearance of normal keratocyte nuclei.5,6 The extra-cellular matrix was highly irregular compared to the nonscarred peripheral area of the same cor-

nea. Some of the images obtained from CM of Patient B contained a mixture of hyper-reflectivity and evidence of epithelial nuclei and are thought to represent images taken from near the apex of the scar. Previous ultrastructural studies have shown the keratoconic stroma to be distorted in regions where there are breaks in Bowman’s layer.16,35,54 Fibrillar degeneration and fibroblastic accumulation have been demonstrated in the stroma beneath these breaks.33 Keratocyte morphology has also been shown to be abnormal in the keratoconic eye.13 These observations are consistent with CM observations in the MKS17–20 of significant abnormalities of keratocyte nuclei, stromal haze and hyper-reflectivity. Hyper-reflective keratocyte nuclei probably represent fibroblasts as observed with the LM. Research into the wound-healing response of the stroma has revealed the presence of hyper-reflective keratocyte nuclei. These have been referred to as activated keratocytes, that is, keratocytes activated to a repair phenotype (or fibroblasts). Using rabbit corneas, Møller© 2007 The Authors



Keratoconus A

C

partly responsible for the transparency of the cornea.59 Møller-Pedersen and colleagues55 suggested that the deposition of a new extra-cellular matrix may also contribute to corneal haze following photorefractive keratectomy.

Stromal nerves Simo Mannion, Tromans and O’Donnell 60 investigated stromal nerve morphology and corneal sensitivity in 13 patients with B keratoconus and 13 age-matched control subjects, using in vivo CM and noncontact (‘air puff’) corneal aesthesiometry. Stromal nerve fibre density was found to be significantly lower in keratoconic patients (1,018 ± 490 µm) versus control subjects (1,821 ± 790 µm) (p = 0.006). The mean diameter of stromal nerve fibres Normal cornea ‘Normal’ keratocyte nuclei was found to be greater in patients with keratoconus (10.2 ± 4.6 µm) compared to Figure 12 A. CM image of hyper-reflective and distorted keratocyte nuclei in Patient B, possibly control subjects (5.5 ± 1.9 µm) (p = 0.007). The orientation of stromal nerve fibres in representing activated fibroblasts. the patients with keratoconus appeared B. CM image of keratocyte nuclei in a normal control subject C. LM of anterior stroma of Patient B. The box indicates a region of distorted keratocyte to be altered from the predominantly vertical orientation seen in control nuclei. Normal keratocytes are present below this region ( ¥ 40 objective). subjects. Corneal touch threshold was similar in the two groups, although corneal sensitivity in patients with keratoconus using contact lens corrections (1.18 ± 0.19 g/mm2) Pedersen and co-workers55 showed that the The extreme levels of stromal haze was reduced (that is, the ‘air puff’ preshaze apparent on CM following photoobserved with the CM cannot be attribsure needed to be higher to elicit a refractive keratectomy was due to the uted solely to an accumulation of fibrosensation) compared to the contact lensincreased reflectivity of migrating and actiblasts. Keratocytes are known to be wearing control subjects (0.98 ± 0.05 g/ vated keratocytes. Similar findings have responsible for the production of the mm2) (p = 0.03). Simmo Mannion and been documented in humans. In the early extracellular matrix and in turn, this colleagues60 concluded that there is a stages of keratocyte activation, the nuclei affects the arrangement of collagen within significant reduction in stromal nerve become more apparent. A more extreme the corneal stroma. LM of the cornea of density in the keratoconic cornea, the wound-healing response results in the cell Patient B revealed the presence of abnorreduced stromal nerve density is a cause 56 bodies becoming visible. Transmission mal collagenous tissue surrounding fibroof the reduced corneal sensitivity in keraelectron microscopy has shown that these blasts. Studies using X-ray diffraction have toconic contact lens wearers and the thickhyper-reflective cells represent keratocytes shown that the normal arrangement of ened stromal nerve fibres observed in activated to a repair phenotype. 57 The collagen fibres is severely disrupted in keratoconic corneas may explain why 17–20 58 MKS demonstrated what appears to be scarred regions of the keratoconic eye. prominent stromal nerves are often seen activation of keratocytes in association This is clearly demonstrated by the histousing SLB in such patients. 61 with apical scarring in the keratoconic eye. logical investigations performed in the Corneal nerves may play an active role 17–20 The apparent association between stromal MKS. The irregular arrangement of in the degenerative changes that occur in haze and the appearance of activated kerathis tissue will contribute significantly to keratoconus, by facilitating keratocytetocytes suggests that keratocyte hyperthe resulting stromal haze observed using epithelial interactions. Brookes and reflectivity may serve as a useful marker of CM as the normal lattice arrangement of associates62 observed nuclei of aberrant disease progression in longitudinal studies the collagen fibres is disrupted. Regular anterior keratocytes wrapping around of keratoconus, which could be monitored arrangement of the collagen fibres nerves as they passed through the otherin vivo using CM. within the corneal stroma is known to be wise acellular Bowman’s layer from the © 2007 The Authors Journal compilation © 2007 Optometrists Association Australia

Clinical and Experimental Optometry 91.1 January 2008 45


stroma to the epithelium. As the keratoconus progressed and Bowman’s layer degraded, these keratocytes were seen to express higher levels of the lysosomal enzymes cathepsin B and G and to become displaced anteriorly into the epithelium. Localised nerve thickening also developed within the epithelium in association with cathepsin B and G expression and appeared to be destructive to the cornea. Specifically, the authors noted that enzyme activity by keratocytes seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowman’s layer and the stroma. Observations of apparent intermixing of epithelial cells, keratocytes from the anterior stroma and nerve fibres within split sections of Bowman’s layer made in the MKS 17–20 using CM and LM, support the keratocyteepithelial interaction theory of Brookes and associates. 62

Striae Alternating dark and light bands were observed with CM in the stromal images of 45 per cent of keratoconic eyes examined in the MKS.17–20 The bands corresponded with the appearance of Vogt’s striae on SLB examination. Figure 13A shows a SLB image of striae visible in a keratoconic patient. When magnified, the image of the striae taken with the SLB (Figure 13B) is strikingly similar to the CM image of bands in the posterior stroma of a keratoconic patient (Figure 13C). Bands observed with the CM were most commonly in the posterior stroma. Posterior bands varied in width, ran mainly in a near vertical direction and appeared to run a straight course through individual image frames. Keratocyte nuclei were located in between the bands but their distribution appeared unaffected by the presence of bands. Nerve fibres appeared to run a straight course through the bands. When present, bands in the anterior stroma showed greater variability in width and direction within a single frame. Bands were present only in the anterior stroma in more severe levels of keratoconus. No obvious correlate of Clinical and Experimental Optometry 91.1 January 2008 46

Figure 13 A. SLB image of a keratoconic cor nea, with striae visible in the optic section B. Magnified image of the striae shown in (A) C. CM image of bands in the posterior stroma of a patient with keratoconus

banding could be observed with the LM (Figure 14). Uçakhan and colleagues25 observed folds, which they referred to as Vogt’s striae, in 50 per cent of the keratoconic eyes they examined. Folds were seen in the anterior stroma in 21 eyes (44 per cent), in the mid-stroma in 21 eyes (44 per cent) and in the posterior stroma in 24 eyes (50 per cent). Interestingly, their description of stromal folds as representing ‘crests and troughs’ suggests that they believe folds to have a three-dimensional construct. For example, Uçakhan and colleagues 25 stated that keratocyte nuclei were visible only over the light bands, which they called ‘crests’, and were not seen on longitudinal dark bands, which they believed corresponded to ‘troughs’. Posterior stromal folds were observed in 14 eyes with severe keratoconus, eight eyes with moderate keratoconus and two eyes with mild keratoconus. In the earlier studies of Wygledowska-Promienska and associates 24 and Somodi and colleagues,23 folds were observed only in the posterior stroma.

The images obtained in the MKS 17–20 suggest that the stromal bands seen with the CM represent collagen lamellae under stress rather than folds. Komai and Ushiki63 demonstrated a differential arrangement of collagen lamellae in the anterior and posterior corneal stroma. Anterior lamellae are 0.5 to 30 µm wide, they have a flat tape-like shape, run in random directions and are often intertwined. The lamellae of the posterior stroma are wider (100 to 200 µm) and have the appearance of broad sheets. 63 The CM images obtained of bands in the anterior and posterior stroma show a similar pattern to those described by Komai and Ushiki. 63 Anterior stromal bands are narrower and irregularly spaced. A tape-like shape was observed in some patients. The pattern of banding varied in sequential frames. Bands in the posterior stroma were wider, regularly spaced and often consistent in direction in several sequential frames. If clinically observed Vogt’s striae represented folds (rather than lines of stress), then their appearance © 2007 The Authors



not be seen in the normal eye because Bowman’s layer is under tension due to intraocular pressure. However, Dangel and Kracher69 observed the mosaic pattern in 75 per cent of eyes of keratoconic patients wearing rigid lenses versus five per cent of non-keratoconic rigid lens wearers. These observations introduce the possibility that the disarrangement of the collagen network in the keratoconic eye somehow facilitates the appearance of a mosaic pattern when the cornea is stressed by the pressure of a rigid lens. Thus, the appearance of dark bands in keratoconic patients in the MKS (50 per cent of whom were wearing rigid lenses) may be, at least Figure 14. Bands observed with CM in keratoconus in part, a manifestation of this mosaic A. Bands of varying width in the anterior stroma, running orthogonally at approximately formation. 90 degrees and 180 degrees The direction of the bands in the posteB. Fine vertical bands in the anterior stroma. A bifurcating nerve fibre and keratocytes rior stroma was found to correlate well are visible. with the steepest Sim-K axis of the cornea C. Vertically oriented bands in the mid-stroma as determined by corneal topography. 18 D. Vertically oriented bands in the mid-stroma with faint horizontal banding Therefore, it seems probable that the oriE. Bands in the posterior stroma, with a nerve fibre crossing horizontally. Keratocytes entation of the bands is due to a pattern are visible only between the dark bands. of stress in the collagen lamellae emerging F. Widely-spaced dark bands in the posterior stroma from the apex of the cone. By way of example, consider Figure 15A, which is a schematic representation of a presumed ‘stress pattern’, templated on top of a topographon CM would not be expected to be Disruption to the arrangement of colical map of a keratoconic cornea, which related to the pattern of collagen lamellae. lagen lamellae at any level of the stroma may emanate from the apex of an inferoThe transparency of the corneal stroma will cause light to be refracted differently, nasally located cone. in the normal eye is, in part, due to the thereby having an effect on the mutual If the objective lens of the CM were to regular and precise arrangement of the interference of light rays passing through be positioned for examination of the cen59 collagen fibrils. Maurice demonstrated the corneal stroma. The CM images contral cornea, an area superior and slightly that for the corneal stroma to be transpartaining stromal bands demonstrate variatemporal to the cone apex would be exament, it is necessary that fibrils are parallel, tions in contrast, presumably due to the ined (Figure 15A, red box). Stress lines equal in diameter and have their axes disdisrupted arrangement and irregular sep- would run through the field of view at posed in a regular lattice formation. This aration of the collagen fibrils. CM images about 80 degrees (using conventional regular arrangement results in mutual of stromal bands in keratoconus do not ophthalmic lens axis notation). This interference of the light rays leading to have the uniformity of images obtained of would result in dark banding as shown in 59 minimal light scattering. This effect is collagen fibrils in the normal cornea. 58,65 Figure 15B. If the objective lens of the CM likely to be greatest at the posterior Both of these findings indicate significant were to be positioned for examination of stroma, as a result of the more regular alterations of the collagen fibre arrangethe apex of the cone (Figure 15A, blue 64 fibril arrangement found posteriorly. ment in the keratoconic eye. This may be box), stress lines might be seen running The banded appearance of the stroma partly responsible for the reduced vision through the field of view at a variety of observed on CM in keratoconic patients in keratoconic patients. angles. This would result in dark banding 68 may represent a widespread, irregular Tripathi and Bron described the as shown in Figure 15C. separation of individual collagen fibrils appearance of a secondary mosaic in the The orientation of banding in the stro within the lamellae. Indeed, a number cornea, the structural basis of which lies in mal images of keratoconic patients ob65–67 served in CM images captured from the of authors has demonstrated marked the particular arrangement of many promabnormalities in the organisation of the inent collagen lamellae of the anterior central cornea are consistent with this anterior corneal collagen lamellae of stroma that take an oblique course to gain schematic model. In many patients, the keratoconic corneas. insertion into Bowman’s layer. This can cones were located inferiorly to the cenAnterior stroma

Mid stroma


Posterior stroma



B

A

C

Nasal

Temporal

Figure 15. Model to illustrate the stress pattern theory of stromal banding observed in keratoconus A. Topographic map of a keratoconic cornea, with ‘stress lines’ emanating from the apex of the cone. The red box indicates the region of central cornea and the blue box the region of the cone, imaged with the CM. B. Expected CM image of the central cornea, with predominantly vertically oriented bands corresponding to stress lines running in that direction C. Expected CM image of the cone, with bands running in all directions

tre of the cornea and the banding was near-vertical. The findings for the patient with a centrally located cone are also consistent with this theory. This patient displayed highly irregular posterior banding, namely, faint bands were apparent horizontally in addition to more prominent vertically orientated bands. This mixed banding pattern indicates that the stress in the cornea corresponds to the apex of the centrally located cone. Further research would need to be undertaken, by way of imaging banding patterns at various locations on keratoconic corneas, to test the hypothesis that banding represents stress lines emanating from the cone apex. X-ray scattering has unambiguously demonstrated that the majority of collagen fibrils in the central cornea adopt a preferred orientation in the inferiorsuperior and nasal-temporal directions. 70 If observations of banding (with the CM) and striae (with the SLB) in the central Clinical and Experimental Optometry 91.1 January 2008 48

cornea are related to the orientation of collagen fibrils, then these formations would be expected to be found horizontally as well as vertically. On examination with CM in the MKS, 17–20 horizontal and vertical bands were observed in the anterior stroma and predominantly vertical striae were observed in the posterior stroma. Vogt’s striae in the same patients seemed to be predominantly oriented vertically when viewed with the SLB. Although the early literature 71 suggests that Vogt’s striae are primarily vertically oriented when observed with the SLB, more recent anecdotal SLB observations of striae in keratoconus patients (Gavin O’Callaghan, personal communication) indicate that striae can occur at any orientation (Figure 16). In addition, based on their observations of photographic images of striae captured from over 1,500 keratoconic patients, senior authors of the CLEK study 21 are of the opinion that striae in keratoconus can occur at any angle (Karla

Zadnik, Joe Barr and Timothy Edrington, personal communication). Smolek and McCarey 72,73 studied the cohesive strength of corneal lamellae across the cornea. Investigations of the lamellae in the vertical meridian have shown that the inferior cornea has the least cohesive strength. 73 Varying patterns of cohesive strength were seen between individuals but paired corneas often display the same strength profiles. Smolek 73 reported a circumstantial correlation between cohesive strength and the patterns seen in the different forms of corneal ectasia. In keratoconus, the cone is most often located inferiorly or centrally, 74 corresponding to the areas of reduced strength found by Smolek. 73 The appearance of stromal banding in the MKS 17–20 may represent a stress-related change in corneal lamellae, corresponding to areas of reduced strength in the cornea and the formation of an ectatic cone-like protrusion in that area. Examination of other ectatic degenerations such as keratoglobus and pellucid marginal degeneration may shed further light on this hypothesis by revealing different patterns of banding corresponding to regions of reduced cohesive strength of stromal lamellae and clinical evidence of ectasia.

Desçemet’s membrane No abnormalities were detected with the CM at the level of Desçemet’s membrane in the MKS;17–20 however, WygledowskaPromienska and associates 24 observed central detachment of the Desçemet’s membrane and the endothelium from the stroma in advanced keratoconus. Uçakhan and colleagues 25 observed folds at the level of Desçemet’s membrane in eight per cent of keratoconic eyes. Using LM, Chi, Katzin and Teng 33 observed folds and buckling at the level of Desçemet’s membrane in the later stages of keratoconus and ruptures were observed in Desçemet’s membrane in 12 per cent of corneas. These defects were filled first with endothelial cells and later with a newly formed membrane. Ruptures in Desçemet’s membrane are thought to be associated with previous cases of cor© 2007 The Authors



Figure 16. SLB photographs from patients with keratoconus A. Oblique or ‘Y-shaped’ striae (arrow) B. Horizontal striae (arrows) (Photograph courtesy Gavin O’Callaghan)

Keratoconus

Normal cornea

Figure 17 A. Elongated endothelial cells in the infer ior right field of a CM image of a patient with keratoconus B. CM image of normal endothelium in a control subject

neal hydrops. That Desçemet’s membrane was normal in the MKS 17–20 is not surprising in view of the absence of a pre vious history of hydrops in any of the patients.

Endothelium The endothelial images obtained from one patient in the MKS 17–20 displayed evidence of elongated cells (Figure 17). This appearance was verified by a masked inde-

pendent observer who was experienced in evaluating CM images. The region of the stroma anterior to these elongated cells and the remainder of the field of the endothelium adjacent to the elongated cells appeared normal. There was no evidence of elongated endothelial cells in any other patient examined with the CM. The mean endothelial cell density (ECD) in keratoconus was six per cent greater than that of normal controls.


Many of the endothelial images seemed to display a large number of smaller cells with only scattered large cells, however, there was no difference in endothelial polymegethism between keratoconic patients (0.35 ± 0.05) and control subjects (0.38 ± 0.07) (paired t-test: t = 1.8, p = 0.08). Pleomorphism and enlarged endothelial cells were seen in 13 per cent of eyes with severe keratoconus by Uçakhan and colleagues.25 In one (two per cent) eye with severe keratoconus with no identifiable history of acute hydrops, folds in Desçemet’s membrane and endothelial guttata were observed. These authors found no difference in mean ECD or mean endothelial cell area between keratoconic patients and controls. In eyes with severe keratoconus, the mean ECD was lower than in eyes with moderate (p < 0.05) or mild (p < 0.05) keratoconus and the mean endothelial cell area was higher than in eyes with mild keratoconus (p < 0.05). The mean endothelial cell hexagonality was lower in keratoconic eyes (p < 0.05). Weed and associates 26 found no difference in ECD between keratoconic patients and control subjects. With three different studies indicating either increased,19 decreased25 or normal26 ECD and either decreased or normal polymegethism in keratoconus, our understanding of the true state of the corneal endothelium in keratoconus remains uncertain. A summary of ECD in keratoconic patients as measured by various authors is presented in Table 3. Early studies with specular microscopy showed that there is a significant increase in the amount of pleomorphism and polymegethism in the keratoconic eye 75,76 however, these studies did not disclose whether the patients examined wore contact lenses. Halibis 77 has shown that the level of polymegethism and pleomorphism in keratoconic patients is similar to that of lens-wearing controls. This finding is consistent with those of the MKS. 17–20 Hoffer and Kraff 78 investigated the endothelium in a large series of normal eyes using specular microscopy and found that ECD was significantly higher in eyes with a longer axial length. Using the same Clinical and Experimental Optometry 91.1 January 2008 49


technique, Esgin and Erda 79 demonstrated an increase in central ECD following wear of high oxygen transmissible rigid lenses. In the majority of cases, myopia and rigid lens-wear are features of keratoconus. This may account for the increased ECD found in the MKS. 17–20 In the MKS,17–20 the endothelial cells of the cornea of Patient B appeared normal when viewed with LM (Figure 18). It was not possible to correlate these findings against those from CM as the endothelium of Patient B was obscured by high levels of haze in the anterior cornea. In the early stages of keratoconus, the endothelium has a normal appearance when viewed with the LM. 33 In more advanced cases, it shows flattening and the nuclei are further apart. 33 Specular microscopy has revealed an increase in pleomorphism and also a high proportion of small endothelial cells in keratoconus. 75 Large elongated cells were also apparent adjacent to the cone, with the long axis of these cells oriented towards the cone apex.75 Such observations are consistent with the notion that corneal tissue is being ‘stretched’ as a result of ectasia. In the MKS,17–20 evidence of endothelial cell elongation was observed in only one patient. The lack of cellular elongation in the majority of the study group may be attributed to the fact that the central cornea (thus typically not the centre of the cone) was imaged in all patients. LM of the endothelium of Patients A and B showed the cells of this layer to be normal in appearance. Endothelial cell degeneration has been reported in corneas with more severe levels of keratoconus, with the damage being more prevalent at the base of the cone rather than at the apex.15 These changes were not observed with the CM in the MKS, 17–20 probably due to the fact that only the central cornea was investigated and the endothelium beneath the cone was often obscured by haze and scarring. KERATOCONUS AND CONCURRENT CORNEAL DISEASE

The CM has been used to examine cases of disease that have occurred in the corClinical and Experimental Optometry 91.1 January 2008 50

Author

Year

Keratoconus

Control

p-value

Hollingsworth, Efron and Tullo19 Uçakhan and colleagues 25 Weed and associates 26

2005 2006 2007

3,250 ± 352b 2,754 ± 312c 2,888 ± 380d,e 2,941 ± 464d,f

3,056 ± 365c 2,900 ± 354c 3,043 ± 264c 3,043 ± 264c

p < 0.05 NSg NSg NSg

a

Units of density are cells/mm 2 b Mixture of lens wearers and non-lens wearers c Only non-lens wearers d Only lens wearers e Moderate keratoconus f Advanced keratoconus g Not significant (p > 0.05)

Table 3. Endothelial cell densitiesa in patients with keratoconus reported by various authors

Endothelial cell nuclei

Figure 18 A. LM section of the cornea of Patient B ( ¥ 10 objective) B. Enlarged LM image of the endothelium of Patient B ( ¥ 20 objective). Arrows indicate nuclei of endothelial cells.

nea of keratoconic patients. Such studies are important because they can provide unique insights into the keratoconic cornea by revealing how this tissue responds to the stress of additional pathology.

Acute hydrops Grupcheva and associates 80 reported the case of a Caucasian man with a history of keratoconus since teenage years. He presented with unusual bilateral keratoconus with acute hydrops that had developed © 2007 The Authors



over a three- to four-month period. The patient had no previous history of contact lens wear. At the time of examination, the patient presented with spectacle visual acuity of 6/60 in both eyes. Typical conical deformation of each cornea was evident with the SLB, with well-circumscribed oedema at the apex of the cone, which was slightly more prominent in the cornea of the right eye. No other pathology was highlighted on clinical examination. Orbscan topography could not be performed as a result of decreased corneal transparency. Accurate intraocular pressure measurement was not possible. Anterior stromal oedema was observed, with marked subepithelial bullae and folds in Desçemet’s membrane. Corneal scarring was present only anteriorly at the level of Bowman’s layer. These in vivo CM findings confirmed the diagnosis of keratoconus with a less common bilateral presentation of acute hydrops.

investigation of the efficacy of these treatments in uncomplicated cases and allows adverse reactions to be studied at a cellular level.

Riboflavin-UVA-induced collagen cross-linking Wollensak, Spoerl and Seiler82 have described the technique of riboflavin/ ultraviolet A (UVA)-induced collagen cross-linking, which is designed to bring the progression of keratoconus to a halt. The underlying theory is that there will be an increase in corneal biomechanical stiffness due to enhanced collagen crosslinking as a result of the treatment. Mazzotta and colleagues 83 assessed corneal tissue modifications using this treatment in a group of 10 patients with progressive keratoconus, as well as regeneration of the epithelium and subepithelial nerve plexus, using the HRT II CM. Treatment included instillation of a 0.1% riboflavin/20% dextran solution five minEpidemic keratoconjunctivitis utes before UVA irradiation and every five 81 Alsuhaibani, Sutphin and Wagoner minutes for a total of 30 minutes thereafter. A dual UVA (370 nm) light-emitting reported the case of a 14-year-old Saudi girl with keratoconus who developed diode was used to generate radiant energy sub-epithelial infiltrates after the onset of 5.4 Joule/cm2. The protocol included of bilateral epidemic keratoconjunctivitis. the operation followed by antibiotic medCM of the left cornea, conducted eight ication and eye dressing with a soft thera weeks after the onset of the infection, peutic contact lens. showed many highly reflective dendritic After five days of soft contact lens wear, cells at the level of the basal epithelium the corneal epithelium displayed a reguand anterior stroma. Many highly relar morphology and density with CM. Disflective fusiform and round cells were appearance of subepithelial stromal observed within the anterior stroma, with nerve fibres was observed in the central decreasing density in progressively deeper irradiated area where initial reinnervalayers of the stroma. These findings were tion was observed microscopically one not present on CM that had been permonth after the operation. No changes in formed two weeks before the onset of nerve fibres were observed in the periphepidemic keratoconjunctivitis. In this case, eral untreated cornea, with a clear lateral CM examination provided clear evidence transition between the two areas. Six of an inflammatory response localised to months after the operation, the anterior the basal epithelium and anterior stroma subepithelial stroma was recolonised by of the central cornea. nerve fibres with restoration of corneal sensitivity. 83 A similar pattern of disappearance and MEDICAL AND SURGICAL regeneration of keratocytes was observed INTERVENTIONS IN KERATOCONUS using CM.84 A reduction in KD in the ante A number of medical and surgical aprior and intermediate stroma and stromal proaches can be applied to the treat- oedema, was observed immediately after ment of keratoconus. The CM facilitates treatment. Keratocytes were observed to © 2007 The Authors Journal compilation © 2007 Optometrists Association Australia

repopulate the central cornea three months after the operation and the oedema had disappeared. At six months post-operatively, keratocyte repopulation was complete. No endothelial damage was observed at any time.

Intrastromal corneal ring implants Intrastromal corneal ring implants are corneal inlays made of plastic, with an arc length of 150 degrees, which are used for the correction of low to moderate myopia. The outward radial tension of these rings leads to a reduction in curvature of the cornea and a normalisation of corneal topography, resulting in reduced myopia, less optical aberration and improved vision. More recent developments of this technique include short arc length segments (130 degrees) for the correction of myopia concurrent with astigmatism and radially-placed corneal inlays for the correction of hyperopia. Ruckhofer and colleagues85 used CM to examine the corneas of a series of keratoconic patients who had implants inserted at a single surgical centre. Weeks and months after implantation, ‘lamellar channel deposits’ regularly appeared around the segments. This material was thought to consist of intracellular lipids. Kymionis and associates 86 examined 17 eyes of 15 patients with keratoconus aged 24 to 52 years (mean: 34 ± 11 years), who had completed five years after insertion of two intrastromal segments of 0.45 mm thickness in the cornea of each eye. When examined using CM, most patients exhibited normal central corneal morphology in all layers, with normal epithelial cells, subepithelial nerve plexus, keratocyte distribution and endothelial morphology. Needle-shaped keratocytes and tortuous sub-basal nerves were observed within the stroma in one patient. Microdeposits, stretched keratocytes and mild fibrosis were observed at or close to the anterior channel of all patients. At the plane of the implant, oval-shaped deposits were observed along the channel. One patient exhibited increased fibrosis or collagen disruption a few microns away from the ring segment. Clinical and Experimental Optometry 91.1 January 2008 51


Epikeratophakia Shi and colleagues87 used CM to study 24 cases of keratoconus from three days to five years after epikeratophakia. The tissue lens was observed to be covered by apparently ‘flattened’ superficial corneal epithelial cells three to four days post-operatively. Epithelial wing and basal cells were also observed but the morphology and arrangement of these cells were irregular with low cell density. The superficial flat epithelial cells appeared normal at one month and the morphology and density of the basal epithelial cells tended to be normal at six months post-operatively. The subepithelial nerve plexus appeared irregular at 18 months but appeared normal two years post-surgery. In the stroma of the tissue lens, keratocytes appeared circular, dot-shaped, rodshaped or reticular. A few normal keratocytes were observed at the periphery of the tissue lens two years post-operatively. At five years, KD in the periphery remained lower than that in the centre of the tissue lens. Stromal nerves appeared in the tissue lens six months after surgery and the quantity of nerves had increased after two years but was still less than normal after five years. There was no change in the stroma and endothelium of the recipient cornea. Penetrating keratoplasty A longitudinal evaluation of four patients who had undergone penetrating keratoplasty was undertaken by Hollingsworth, Efron and Tullo 20 for 12 months after surgery, using slit scanning CM. The procedure was preformed because of keratoconus (two patients), Fuch’s dystrophy and lattice dystrophy. Patients were examined on four occasions over a 12-month period after surgery. The epithelium varied in appearance between patients and took at least 12 months to appear normal. Bowman’s layer was viewed as an acellular layer immediately after surgery with no evidence of nerve fibres, although some nerve components were apparent 12 months after surgery. Stromal nerves were not visible immediately after surgery. One year following penetrating keratoplasty, there was Clinical and Experimental Optometry 91.1 January 2008 52

evidence of thin nerves running a straight course through the central stroma. AKD and PKD were lower in the transplanted cornea and appeared to remain constant over a period of 12 months. Activated keratocytes were seen in the anterior stroma of all patients. They appeared to be responsible for significant levels of corneal haze. The time within which this keratocyte activation occurred varied between individuals. ECD decreased at an accelerated rate over the 12-month period. Imre, Resch and Nagymihaly 88 examined seven eyes with clear grafts at 15 and 66 months after penetrating keratoplasty. The preoperative diagnoses were keratoconus (two), granular corneal dystrophy (two), pseudophakic bullous keratopathy (two) and corneal ulcer (one). Mean density of basal epithelial cells was 3,928 ± 378 cells/mm2 at 15 months and 3,284 ± 565 cells/mm2 at 66 months post-operatively. At 15 months, AKD and PKD were 750 ± 113 and 601 ± 98 cells/mm2, respectively, and at 66 months these measures were 383 ± 53 and 411 ± 98 cells/mm2, respectively. ECD decreased from 1719 ± 576 cells/mm2 at 15 months to 965 ± 272 cells/mm2 at 66 months. The results of this study are consistent with the findings of the MKS.17–20 Both were longitudinal evaluations and suggest that there is an ongoing decline in the cellular integrity of corneal grafts up to six years following penetrating keratoplasty. Niederer and colleagues89 conducted a cross-sectional CM study comparing corneas from 42 patients after penetrating keratoplasty with those of 30 controls. Patients were assessed by ophthalmic history, clinical examination and computerised corneal topography. Time after surgery ranged from one month to 40 years (mean: 85 ± 105 months). Significant reductions in epithelial CD (p < 0.001), KD (p < 0.001) and endothelial CD (p < 0.001) were noted in comparison with control corneas. Significant reductions in sub-basal nerve fibre density (p < 0.001) and nerve branching (p < 0.001) were also noted. ECD decreased (r = -0.472; p = 0.003) and nerve fibre density increased (r = 0.328; p = 0.034) with time after

surgery. As an indication for transplantation, keratoconus was associated with higher sub-basal nerve fibre densities (p = 0.003) than other indications for corneal transplantation. Neither nerve fibre nor cell density was correlated with visual acuity. The authors concluded that profound reductions in cell density at every level of the transplanted cornea and alterations to the sub-basal plexus are apparent up to 40 years after penetrating keratoplasty. Forseto Ados and associates 90 reported the case of a 36-year-old female patient with keratoconus who suffered epithelial in-growth after penetrating keratoplasty. This patient had developed a welldelimited posterior hazy membrane covering the inferior two thirds of the cornea three months after an uneventful penetrating keratoplasty. A posterior corneal line was present resembling an endothelial graft rejection line but with no keratic precipitates or corneal oedema. Ocular hypertension was not observed. CM showed the epithelium and stroma to be normal. Two distinct cell types were presented at the endothelium layer. Enlarged endothelial cells were observed in the superior part of the cornea up to the leading edge of the hazy membrane. In the middle and inferior part of the graft, the cells were larger with polygonal shape and easily recognisable hyper-reflective nuclei, suggestive of epithelial cells. The patient promptly received another penetrating graft and histologic analysis confirmed the diagnosis of epithelial in-growth. CONCLUSIONS

This review has illustrated how the CM can be used to identify and evaluate morphological alterations to the corneal epithelium, sub-basal nerve plexus, Bowman’s layer, stroma, Desçemet’s membrane and endothelium in keratoconus. The results of morphometric analyses by various authors are equivocal, so further studies will be required to accurately determine quantitative cellular characteristics of the keratoconic cornea. Pathological changes observed using CM in the corneas of patients with keratoconus could serve as a basis for clinical © 2007 The Authors



y p o c s ) o r o c r t i i v m n n i ( o r t c e l E

l a i c fi r e p u s f o . s r a r e e y p l . p a a o e s w n i a d t r b d r o n e m a n e e t o m t a r s e e n n m e e o m g c e e e s a . d b b d e t s l m l r l a o u e i p c l m e . r e l h o r a t l s t n s i l a p e b o B E c A N

. . . e n s d e u t e o y s i n s t i n a c t l i o h e u t t v m a r i y d t u e l k r e c c a t e c f l o u a r n n a g f t . n e o o e s i c a r i r e t r l , l o i a y r s b v a i d p t l t s o r n , l s c a m a b ’ fi . u b n a d s t d a d e k i l o n n a e a a r n a m h g e w e i t r l b i k n o y a p , o B g o s d e e i i t n l o u a i h m n r t a h s i e s s s n w r i n k p e r i e a o o d t e b t e s g e r m fi a l u t l d b l a fi o n n r h e e s n m e l a t g m a g k l e r o a g a l a i l r l n n e l o a r r o b i e b C F B c F b A

s l . l e d c e l n a s e t a t b a f fl o r e a e c p n a p a a . a r . g s a e t n l i l p n . e n e d c p a r n l a e i t r p r h t a p u a n a o l p l d i m t e a r i v u o r e d l n t v n o s o O I C i N

a . s t n . i s d r a e a h l a t . c i b d a . s s n o w e r f i r u r a o e b u c l r fi o t s n c f e a . p r i o u r u o e a a g n o p r . e e e e n a d n r t m o u i n y i h s t n i e s a a l s l o n c i o n i t c a u i g t n t . n a i e d m u r i o a r a l e u n l e c k m a s r t a e k c c u p i o t c t u y g f n r s n a b e a a c e r l r g e o r , r s e m u e r s t i y g g y o n a d a r e l a i l s e e d l i a r r n o r i B F I b D D K a F

a

y p o c ) s o o t r r i c v i m n t i ( h g i L

y p o c s ) o r o c r i t i v m l n i a c ( o f n o C

a . a . d a i e . l e p i e c a l h c u n s - u e n l e l b i d d s n e i i v p c s a p h r s i - t o y w l r d a r e l l a t a u u g g g n e e r r o i r i r l e , ; : e y s g z l l r a e l h c a : l : s s a l i l l l c e e c fi c r l e g a p i n s u a S W B

l a e r n e r y o a C L

a

. . e e . v c l e i t n b e c a s r i e s a i a fl b e v e r p s . e p e w h r t a e e m f i t i p v o n t y e e f h r n m o u i d o d n t m o s l a n o s e r o . fi e s e p t c p d e , y h a e n d c t h a h t . e . s t t n o y r b y u a e w l t a k . i o s k r l o l e a v l . c k d e e i u r l i a c i t o d r h n z h c g f t r e t a a e c o r / n h t s n r d fl i e e a e i n i f a l a e d p r n o d c e t v r l n n l e a a n o h c u e v p u g s r e u c h l v i t y n l i b . n s l s l e c h e u e e e e e c e g r n r r l r d fl t n b o s b a b e e d y i i fi s r fi c e fi l c t a - n a l o e a r e e d e h e e e t n r e v v v h a t r r t n r i r c p z r e t e f u e p e n y a e l N o B N E N I H H K A

l F H

. n e e s e b t o n n a C

g n i r r a c s l a . i c e z fi a r h e p d u n S a

m u i l e h t i p E

s l u x a l s e a p b - e b v u r e S n

s ’ n a m r w e o y a B l

y p ) . p o c o g s r n m t i a o r r i l v t c i i n r e l i h S m ( c . o i s e i b e z a

d e m r o f y l w e n d n a s l l e c l a i l e h t o d n e h t i w l l . fi e s n a e r r b u t m p e u R m

. l a m r o N

. n o i t a r e n e g e d l l e c l a i l e h t o d n E r e h t r u f i e l c u n d n a g n i n e t t a a . fl t l r l e a p C a

. s l l e c l a i l e h t o d n e d e t a g n o l e f o e c n e d i v e d e t i m i L

. e z a h . d e n a a i r . t g a s n i m s ’ e t r g r a d o c e V S O

e h t f . s e n o ’ i t n y e t s i . a r l p m m e b a o r i ç m m r d x s y o r e e o H p D m N

a m o r t S

s e ’ t n e a r m e b ç m s e e D m


m u i l e h t o d n E

. s u n o c o t a r e k e r e v e S a

s e u q i n h c e t l a n o i t a v r e s b o s u o i r a v g n i s u s u n o c o t a r e k n i s r e y a l l a e n r o c f o e c n a r a e p p A . 4 e l b a T

decision-making. For example, it might be possible to detect changes in epithelial cell size or dark lines (striae) in the stroma, very early in the disease process, providing early detection for keratoconus. The grading scale for stromal haze could be adopted as a basis for classifying or categorising the severity of keratoconus. Evidence of splitting or substantial disruption to Bowman’s layer could serve as a criterion for undertaking penetrating keratoplasty. The appearance of the corneal layers in keratoconus with the CM, compared with that observed with the SLB, LM and electron microscopy, is summarised in Table 4. CM images of keratoconic corneas in vivo have also been correlated with images obtained from in vitro samples of keratoconic corneas using the more established technique of LM. This work provides a framework against which cellular changes in keratoconus can be studied using CM in a clinical context. CM has facilitated a greater understanding of medical and surgical approaches that can be applied to the treatment of keratoconus. The extent of tissue compromise can be assessed at a cellular level to aid diagnosis and can be used to evaluate the recovery process and the efficacy of different treatment interventions. REFERENCES

1. Rabinowitz YS. Keratoconus. Surv Ophthal- mol 1998; 42: 297–319. 2. Chopra I Jain AK. Between eye asymmetry in keratoconus in an Indian population. Clin Exp Optom 2005; 88: 146–152. 3. Haque S Simpson T Jones L. Corneal and epithelial thickness in keratoconus: a comparison of ultrasonic pachymetry, Orbscan II and optical coherence tomography. J Refract Surg 2006; 22: 486–493. 4. Patel DV McGhee CN. Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review. Clin Exp Ophthalmol 2007; 35: 71–88. 5. Efron N Perez-Gomez I Mutalib HA Hollingsworth J. Confocal microscopy of the normal human cornea. Contact Lens Ant Eye 2001; 24: 16–24. 6. Hollingsworth J Perez-Gomez I Mutalib HA Efron N. A population study of the normal cornea using an in vivo , slitscanning confocal microscope. Optom Vis Sci 2001; 78: 706–711. ,

,

,

,

,

,

,

,

,

,



7. Zhivov A Stave J Vollmar B Guthoff R. In vivo confocal microscopic evaluation of Langerhans cell density and distribution in the corneal epithelium of healthy volunteers and contact lens wearers. Cornea 2007; 26: 47–54. 8. Patel DV McGhee CN. Mapping of the normal human corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy. Invest Ophthalmol Vis Sci 2005; 46: 4485–4488. 9. Böhnke M Masters BR. Long-term contact lens wear induces a corneal degeneration with microdot deposits in the corneal stroma. Ophthalmology 1997; 104: 1887– 1896. 10. Parmar DN Awwad ST Petroll WM Bowman RW McCulley JP Cavanagh HD. Tandem scanning confocal corneal microscopy in the diagnosis of suspected acanthamoeba keratitis . Ophthalmology 2006; 113: 538–547. 11. Brasnu E Bourcier T Dupas B Degorge S Rodallec T Laroche L Borderie V Baudouin C. In vivo confocal microscopy in fungal keratitis. Br J Ophthalmol 2006; 91: 588–591. 12. Krachmer JH Feder RS Belin MW. Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol 1984; 28: 293–322. 13. Teng CC. Electron microscope study of the pathology of keratoconus: I. Am J Ophthal- mol 1963; 55: 18–47. 14. Scroggs MW Proia AD. Histopathological variation in keratoconus. Cornea 1992; 11: 553–559. 15. Sturbaum CW Peiffer RL Jr. Pathology of corneal endothelium in keratoconus. Oph- thalmologica 1993; 206: 192–208. 16. Sawaguchi S Fukuchi T Abe H Kaiya T Sugar J Yue BY. Three-dimensional scanning electron microscopic study of keratoconus corneas. Arch Ophthalmol 1998; 116: 62–68. 17. Hollingsworth JG Bonshek RE Efron N. Correlation of the appearance of the keratoconic cornea in vivo by confocal microscopy and in vitro by light microscopy. Cornea 2005; 24: 397–405. 18. Hollingsworth JG Efron N. Observations of banding patterns (Vogt striae) in keratoconus: a confocal microscopy study. Cornea 2005; 24: 162–166. 19. Hollingsworth JG Efron N Tullo AB. In vivo corneal confocal microscopy in keratoconus. Ophthalmic Physiol Opt 2005; 25: 254– 260. 20. Hollingsworth JG Efron N Tullo AB. A longitudinal case series investigating cellular changes to the transplanted cornea using confocal microscopy. Contact Lens Ant Eye 2006; 29: 135–141. 21. Zadnik K Barr JT Gordon MO Edrington TB. Biomicroscopic signs and disease severity in keratoconus. Collaborative Longitudi,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,


nal Evaluation of Keratoconus (CLEK) Study Group. Cornea 1996; 15: 139–146. 22. Perez-Gomez I Hollingsworth J Efron N. Effects of benoxinate hydrochloride 0.4% on the morphological appearance of the cornea using confocal microscopy. Contact Lens Ant Eye 2004; 27: 45–48. 23. Somodi S Hahnel C Slowik C Richter A Weiss DG Guthoff R. Confocal in vivo microscopy and confocal laser-scanning fluorescence microscopy in keratoconus. Ger J Ophthalmol 1996; 5: 518–525. 24. Wygledowska-Promienska D Rokita-Wala I Gierek-Ciaciura S Piatek-Koronowska G. The alterations in corneal structure at III/ IV stage of keratoconus by means of confocal microscopy and ultrasound biomicroscopy before penetrating keratoplasty. Klin Oczna 1999; 101: 427–432. 25. Uçakhan OO Kanpolat A Ylmaz N Özkan M. In vivo confocal microscopy findings in keratoconus. Eye Contact Lens 2006; 32: 183– 191. 26. Weed KH Macewen CJ Cox A McGhee CN. Quantitative analysis of corneal microstructure in keratoconus utilising in vivo confocal microscopy. Eye 2007; 21: 614–623. 27. Tsubota K Mashima Y Murata H Sato N Ogata T. Corneal epithelium in keratoconus. Cornea 1995; 14: 77–83. 28. Mocan MC Irkec M. Fluorescein enhanced confocal microscopy in vivo for the evaluation of corneal epithelium. Clin Experiment Ophthalmol 2007; 35: 38–43. 29. Rokita-Wala I Gierek-Ciaciura S Majlinger R Wygledowska-Promienska D. Iron deposits in cornea in confocal microscope. Klin Oczna 2001; 103: 111–115. 30. Scroggs MW Proia AD. Histopathological variation in keratoconus. Cornea 1992; 11: 553–539. 31. Aktekin M Sargon MF Cakar P Celik HH Firat E. Ultrastructure of the cornea epithelium in keratoconus. Okajimas Folia Anat Jpn 1998; 75: 45–53. 32. Jongebloed WL Worst JF. The keratoconus epithelium studied by SEM. Doc Ophthalmol 1987; 67: 171–181. 33. Chi HH Katzin HM Teng CC. Histopathology of keratoconus. Am J Ophthalmol 1956; 42: 847–860. 34. Tsubota K. In vivo observation of the corneal epithelium. Scanning 1994; 16: 295– 299. 35. Tsubota K Yamada M Naoi S. Specular microscopic observation of human corneal epithelial abnormalities. Ophthalmology 1991; 98: 184–191. 36. Patel DV McGhee CN. Mapping the corneal sub-basal nerve plexus in keratoconus by in vivo laser scanning confocal microscopy. Invest Ophthalmol Vis Sci 2006; 47: 1348–1351. 37. Patel DV McGhee CN. Contemporary in vivo confocal microscopy of the living ,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

human cornea using white light and laser scanning techniques: a major review. Clin Experiment Ophthalmol 2007; 35: 71–88. 38. Szaflik JP. Comparison of in vivo confocal microscopy of human cornea by white light scanning slit and laser scanning systems. Cornea 2007; 26: 438–445. 39. Auran JD Koester CJ Rapaport R Florakis GJ. Wide field scanning slit in vivo confocal microscopy of flattening-induced corneal bands and ridges. Scanning 1994; 16: 182– 186. 40. Kobayashi A Yokogawa H Sugiyama K. In vivo laser confocal microscopy of Bowman’s layer of the cornea. Ophthalmology 2006; 113: 2203–2208. 41. Kaas-Hansen M. The histopathological changes of keratoconus. Acta Ophthalmol Scand 1993; 71: 411–414. 42. Fini ME. Keratocyte and fibroblast phenotypes in the repairing cornea. Prog Retin Eye Res 1999; 18: 529–551. 43. Patel SV McLaren JW Hodge DO Bourne WM. Confocal microscopy in vivo in corneas of long-term contact lens wearers. Invest Ophthalmol Vis Sci 2002; 43: 995– 1003. 44. Erie JC Patel SV McLaren JW Nau CB Hodge DO Bourne WM. Keratocyte density in keratoconus. A confocal microscopy study. Am J Ophthalmol 2002; 134: 689–695. 45. Jalbert I Stapleton F. Effect of lens wear on corneal stroma: preliminary findings. Aust NZ J Ophthalmol 1999; 27: 211–213. 46. Efron N Mutalib HA Perez-Gomez I Koh HH. Confocal microscopic observations of the human cornea following overnight contact lens wear. Clin Exp Optom 2002; 85: 149– 155. 47. Efron N Perez-Gomez I Morgan PB. Confocal microscopic observations of stromal keratocytes during extended contact lens wear. Clin Exp Optom 2002; 85: 156–160. 48. Kallinikos P Morgan P Efron N. Assessment of stromal keratocytes and tear film inflammatory mediators during extended wear of contact lenses. Cornea 2006; 25: 1– 10. 49. Kallinikos P Efron N. On the etiology of keratocyte loss during contact lens wear. Invest Ophthalmol Vis Sci 2004; 45: 3011–3020. 50. Hollingsworth JG Efron N. Confocal microscopy of the corneas of long-term rigid contact lens wearers. Contact Lens Ant Eye 2004; 27: 57–64. 51. Wilson SE He YG Weng J Li Q McDowall AW Vital M Chwang EL. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 1996; 62: 325–327. 52. Kim WJ Rabinowitz YS Meisler DM Wilson SE. Keratocyte apoptosis associated with keratoconus. Exp Eye Res 1999; 69: 475–481. ,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,



53. Fabre EJ Bureau J Pouliquen Y Lorans G. Binding sites for human interleukin 1 alpha, gamma interferon and tumor necrosis factor on cultured fibroblasts of normal cornea and keratoconus. Curr Eye Res 1991; 10: 585–592. 54. Tuori AJ Virtanen I Aine E Kalluri R Miner JH Uusitalo HM. The immunohistochemical composition of corneal basement membrane in keratoconus. Curr Eye Res 1997; 16: 792–801. 55. Møller-Pedersen T Li HF Petroll WM Cavanagh HD Jester JV. Confocal microscopic characterization of wound repair after photorefractive keratectomy. Invest Ophthalmol Vis Sci 1998; 39: 487–501. 56. Møller-Pedersen T Cavanagh HD Petroll WM Jester JV. Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy: a 1-year confocal microscopic study. Ophthalmology 2000; 107: 1235–1245. 57. Ohno K Mitooka K Nelson LR Hodge DO Bourne WM. Keratocyte activation and apoptosis in transplanted human corneas in a xenograft model. Invest Ophthalmol Vis Sci 2002; 43: 1025–1031. 58. Daxer A Fratzl P. Collagen fibril orientation in the human corneal stroma and its implication in keratoconus. Invest Ophthal- mol Vis Sci 1997; 38: 121–129. 59. Maurice DM. The structure and transparency of the cornea. J Physiol 1957; 136: 263– 286. 60. Simo Mannion L Tromans C O’Donnell C. An evaluation of corneal nerve morphology and function in moderate keratoconus. Contact Lens Ant Eye 2005; 28: 185– 192. 61. Simo Mannion L Tromans C O’Donnell C. Corneal nerve structure and function in keratoconus: A case report. Eye Contact Lens 2007; 33: 106–108. 62. Brookes NH Loh IP Clover GM Poole CA Sherwin T. Involvement of corneal nerves in the progression of keratoconus. Exp Eye Res 2003; 77: 515–524. 63. Komai Y Ushiki T. The three-dimensional organization of collagen fibrils in the human cornea and sclera. Invest Ophthalmol Vis Sci 1991; 32: 2244–2258. 64. Freund DE McCally RL Farrell RA Cristol SM L’Hernault NL Edelhauser HF. Ultrastructure in anterior and posterior stroma of perfused human and rabbit corneas. Relation to transparency. Invest Ophthalmol Vis Sci 1995; 36: 1508–1523. 65. Morishige N Wahlert AJ Kenney MC Brown DJ Kawamoto K Chikama T Nishida T Jester JV. Second-harmonic imaging microscopy of normal human and keratoconus cornea. Invest Ophthalmol Vis Sci 2007; 48: 1087–1094. 66. Stachs O Bochert A Gerber T Koczan D Thiessen HJ Guthoff RF. The extracellular ,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

matrix structure in keratoconus. Ophthalm- ologe 2004; 101: 384–389. 67. Hayes S Boote C Tuft SJ Quantock AJ Meek KM. A study of corneal thickness, shape and collagen organisation in keratoconus using videokeratography and X-ray scattering techniques. Exp Eye Res 2007; 84: 423–434. 68. Tripathi RC Bron AJ. Secondary anterior crocodile shagreen of Vogt. Br J Ophthalmol 1975; 59: 59–63. 69. Dangel ME Kracher GP Stark WJ. Anterior corneal mosaic in eyes with keratoconus wearing hard contact lenses. Arch Ophthal- mol 1984; 102: 888–890. 70. Meek KM Blamires T Elliott GF Gyi TJ Nave C. The organisation of collagen fibrils in the human corneal stroma: a synchrotron X-ray diffraction study. Curr Eye Res 1987; 6: 841–846. 71. Katz HH. A hypothesis for the formation of vertical corneal striae as observed in the wearing of softlens contact lenses and in keratoconus. Am J Optom Physiol Opt 1976; 53: 420–421. 72. Smolek MK McCarey BE. Interlamellar adhesive strength in human eyebank corneas. Invest Ophthalmol Vis Sci 1990; 31: 1087–1095. 73. Smolek MK. Interlamellar cohesive strength in the vertical meridian of human eye bank corneas. Invest Ophthalmol Vis Sci 1993; 34: 2962–2969. 74. McMahon TT Robin JB Scarpulla KM Putz JL. The spectrum of topography found in keratoconus. CLAO J 1991; 17: 198–204. 75. Laing RA Sandstrom MM Berrospi AR Leibowitz HM. The human corneal endothelium in keratoconus: A specular microscopic study. Arch Ophthalmol 1979; 97: 1867–1869. 76. Matsuda M Suda T Manabe R. Quantitative analysis of endothelial mosaic pattern changes in anterior keratoconus. Am J Oph- thalmol 1984; 98: 43–49. 77. Halabis JA. Analysis of the corneal endothelium in keratoconus. Am J Optom Physiol Opt 1987; 64: 51–53. 78. Hoffer KJ Kraff MC. Normal endothelial cell count range. Ophthalmology 1980; 87: 861–866. 79. Esgin H Erda N. Endothelial cell density of the cornea during rigid gas permeable contact lens wear. CLAO J 2000; 26: 146– 150. 80. Grupcheva CN Craig JP Sherwin T McGhee CN. Differential diagnosis of corneal oedema assisted by in vivo confocal microscopy. Clin Experiment Ophthalmol 2001; 29: 133–137. 81. Alsuhaibani AH Sutphin JE Wagoner MD. Confocal microscopy of subepithelial infiltrates occurring after epidemic keratocon junctivitis. Cornea 2006; 25: 778–780.


,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

82. Wollensak G Spoerl E Seiler T. Riboflavin/ ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Oph- thalmol 2003; 135: 620–627. 83. Mazzotta C Traversi C Baiocchi S Sergio P Caporossi T Caporossi A. Conservative treatment of keratoconus by riboflavinUVA-induced cross-linking of corneal collagen: qualitative investigation. Eur J Ophthalmol 2006; 16: 530–535. 84. Mazzotta C Balestrazzi A Traversi C Baiocchi S Caporossi T Tommasi C Caporossi A. Treatment of progressive keratoconus by riboflavin-UVA-induced cross-linking of corneal collagen: Ultrastructural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans. Cornea 2007; 26: 390–397. 85. Ruckhofer J. Clinical and histological studies on the intrastromal corneal ring segments (ICRS(R), Intacs(R)). Klin Monatsbl Augenheilkd 2002; 219: 557–574. 86. Kymionis GD Siganos CS Tsiklis NS Anastasakis A Yoo SH Pallikaris AI Astyrakakis N Pallikaris IG. Long-term follow-up of Intacs in keratoconus. Am J Ophthalmol 2007; 143: 236–244. 87. Shi W Xie L Li S Yuan F Liu H. Observation on healing process of corneal lens after epikeratophakia with confocal microscopy. Zhonghua Yan Ke Za Zhi 2002; 38: 295–297. 88. Imre L Resch M Nagymihaly A. In vivo confocal corneal microscopy after keratoplasty. Ophthalmologe 2005; 102: 140–146. 89. Niederer RL Perumal D Sherwin T McGhee CN. Corneal innervation and cellular changes after corneal transplantation: an in vivo confocal microscopy study. Invest Ophthalmol Vis Sci 2007; 48: 621–626. 90. Forseto Ados S Dos Santos MS Sampaio A Mascaro V Nosé W. Diagnosis of epithelial ingrowth after penetrating keratoplasty with confocal microscopy. Cornea 2006; 25: 1124–1127. ,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

Corresponding author: Professor Nathan Efron School of Optometry and Institute of Health and Biomedical Innovation Queensland University of Technology Kelvin Grove QLD 4059 AUSTRALIA E-mail: [email protected]

,


keratocon

Recommend Documents