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Clinical and Experimental Optometry 91.1 January 2008

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Journal compilation © 2007 Optometrists Association Australia

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

OPTOMETRY

Clin Exp Optom 2008; 91: 1: 34–55 DOI:10.1111/j.1444-0938.2007.00195.x

Key words: confocal microscope, cornea, keratoconus, keratocyte

Up to the end of the 20th Century, ourclinical view of tissue compromise in liv-ing patients with corneal disease wasrestricted to what could be observedmacroscopically and under magnificationof up to ×40 using a slitlamp bio-microscope (SLB). In the case of keratoconus, the SLB serves as aninvaluable tool for examining gross tissue

changes such as apical scarring, Vogt’sstriae, Fleischer ring (iron depositsaround the base of the cone), cornealthinning, protrusion and hydrops.1 Othertechniques such as retinoscopy,2 cornealtopographic analysis,3 pachometry 3 andoptical 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 ophthalmicclinical landscape, allowing eye-care prac-titioners to non-invasively view the livinghuman 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-

Nathan Efron* BScOptom PhD DSc Joanna G Hollingsworth† BSc(Hons) PhDMCOptom*Institute of Health and BiomedicalInnovation and School of Optometry,Queensland University of Technology,Brisbane, Australia†Optometry Giving Sight, Association ofOptometrists, London, United KingdomE-mail: [email protected]

Confocal microscopy (CM) of keratoconus is reviewed. In the Manchester KeratoconusStudy (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 thedifferences between studies of cell counts is confounded by the use of different experi-mental controls. A consensus exists among studies with respect to qualitative observa-tions. 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 epithelialcells are flattened. Bowman’s layer is disrupted and split in the region of the coneand intermixed with epithelial cells and stromal keratocytes. Stromal haze and hyper-reflectivity observed with CM correspond with apical scarring seen with the slitlampbiomicroscope (SLB). Hyper-reflective keratocyte nuclei are thought to indicate thepresence of fibroblastic cells. Increased haze detected with CM is found with LM to be

due to fibroblastic accumulation and irregular collagen fibres. Dark stromal bandsobserved with CM correlate with the appearance of Vogt’s striae with SLB. Desçemet’smembrane appears normal with both CM and LM. Some evidence of endothelial cellelongation is observed with CM. The application of CM to ophthalmic practice hasfacilitated a greater understanding of medical and surgical approaches that are used totreat keratoconus. This review offers new perspectives on keratoconus and provides aframework, against which tissue changes in this visually debilitating condition can bestudied in a clinical context in vivo using CM.

New perspectives on keratoconus as revealed by

corneal confocal microscopy

Submitted: 1 May 2007Revised: 20 June 2007

Accepted for publication: 6 July 2007

INVITED REVIEW

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Corneal confocal microscopy of keratoconus Efron and Hollingsworth


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lium—the CM allows the cornea to beobserved in vivo at a cellular level.4 Spe-cifically, it is possible to observe individ-ual cells and cell nuclei in the variouslayers of the epithelium and endothe-lium,5,6 cell borders and nuclei of stromalkeratocytes, Langerhans cells,7 the fineepithelial sub-basal nerve plexus8 andnewly-discovered features such as stro-mal ‘microdots’.9 Pathogens such as

acanthamoeba 10

and fungi11

can also beseen in diseased eyes. This technology isallowing researchers and clinicians toembark on a new journey of discovery that is resulting in a more completeand deeper understanding of the livinghuman cornea in health and disease andmay pave the way towards the develop-ment of new medical and surgicalapproaches to the treatment of cornealdisease.

The human cornea can manifest in a variety of abnormal shapes (Figure 1).This review will focus on research that hasemployed CM to characterise the clinicalhistopathology of the keratoconic cornea.Keratoconus is classically consideredas an asymmetrical, progressive, non-inflammatory disorder causing an axialcorneal ectasia. It is characterised by stromal thinning and corneal steepening,leading to irregular astigmatism and myo-pia, which cause a marked distortion in

vision.1,12 Ultrastructural studies13–16 have

demonstrated tissue pathology at all levelsof the keratoconic cornea.

This paper will review recent studiesthat have used CM to examine patientssuffering from keratoconus, to construct acomprehensive model of the ‘clinical his-topathology’ of the living keratoconic cor-nea. Consideration will also be given tothe use of CM to develop a greater under-standing of the keratoconic cornea with

concurrent disease and medical and surgi-cal interventions for keratoconus.

THE MANCHESTER KERATOCONUS

STUDY

The Manchester Keratoconus Study (MKS),17–20 which will form the corner-stone of this review, is a recently com-pleted examination of 29 keratoconicpatients (mean age 31 ± 10 years; range 16to 49 years), who presented consecutively to the outpatient clinic of the Royal EyeHospital in Manchester, UK. Some pa-tients were new to the clinic (referred fora definitive diagnosis) and some wereexisting patients. Both eyes of all patients

were examined, however, due to threepatients having unilateral disease and afurther four patients having previously un-dergone penetrating keratoplasty, data

were obtained from 51 eyes.The mean age at diagnosis, as reported

by the patients, was 21 ± 8 years (range 4

to 42 years). Male patients made up 76 percent of the study group. A family history of keratoconus was present in 17 per cent of patients. The majority of patients (62per cent) were Caucasian, 24 per cent wereof Asian descent and 14 per cent were Afro-Caribbean. Ninety per cent of patientshad bilateral keratoconus and 52 per cent reported that they suffered from someform of atopy, such as asthma, eczema,

hay fever, general allergies or a combina-tion of these. One patient also sufferedfrom retinitis pigmentosa. There were noother associated systemic conditions. A large number of patients reported a his-tory of eye rubbing (66 per cent).

Fifty per cent of eyes were currently fit-ted with rigid contact lenses, the majority of which showed some level of apicaltouch (64 per cent). Thirty-three per cent of eyes were corrected with spectacles.One patient was fitted bilaterally withscleral contact lenses. Three patients wereawaiting surgery and were no longer usingany form of visual correction.

Clinical investigative techniques

SLITLAMP BIOMICROSCOPY

All eyes were examined with a SLB and 27per cent of eyes displayed corneal stain-ing, 37 per cent of eyes demonstrated cor-neal scarring and 37 per cent of eyes had

Vogt’s striae.

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

Cornea plana Normal cornea Keratoconus Keratoglobus

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CORNEAL TOPOGRAPHY

Corneal topography was attempted onall 51 eyes using the EyeSys 2000 Cor-neal Analysis System (EyeSys Technolo-gies, Houston, Texas, USA). This

enabled disease severity to be classifiedby corneal curvature, using the same sys-tem as used in the Collaborative Longi-tudinal Evaluation of Keratoconus(CLEK) study.21 According to this system,disease severity is classified with respect to the curvature of the steepest cornealmeridian as follows: mild, less than 45 D;moderate, 45 to 52 D; and severe, morethan 52 D.

Corneal topographical maps were pro-duced from 46 eyes. In the remainingfive eyes, the cornea was too distorted or

scarred to obtain a reading of corneal curvature; these were classified as severe kera-toconus. The average steep keratometricreading was 52.1 ± 7.9 D (range 42.5 to81.0 D). The majority of patients hadeither moderate or severe keratoconus;specifically, 12 per cent of eyes exhibitedmild keratoconus, 39 per cent of eyes hadmoderate keratoconus and 49 per cent of eyes were classified as having severekeratoconus.

CONFOCAL MICROSCOPY All keratoconic patients and control subjects were examined using an in vivo slit scanning real time CM (Tomey ConfoscanP4, Erlangen, Germany) fitted with an

Achroplan 40X/0.75NA immersionobjective. One drop of local anaesthetic(Benoxinate Hydrochloride 0.4%, Chau-

vin Pharmaceuticals, Romford, UK) wasinstilled into the lower fornix of theeye.22 A drop of polymer gel (Viscotears,CIBA Vision, Duluth, Georgia, USA) wasapplied to the microscope probe prior tothe examination to optically couple themicroscope objective lens to the cornea.CM was performed on the central corneaof all eyes.

Images obtained of the corneal layers of the 51 keratoconic eyes during CM exam-inations were stored on S-VHS videotape.Each examination was evaluated frameby frame by a single examiner. Images

were saved of the epithelium, Bowman’slayer, the anterior and posterior stroma,

Desçemet’s membrane (where visible)and the endothelium. In the course of scanning through the cornea along thecentral anterior-posterior axis, imageframes of the anterior stroma were taken

to be those immediately posterior toBowman’s layer and image frames of theposterior stroma were taken to be thoseimmediately anterior to the endothelium.In addition, images showing any abnor-mality were saved regardless of their posi-tion within the cornea.

For quantitative analysis, only the right eye of each study participant was exam-ined; however, in cases where it was not possible to use the right eye (such as inunilateral keratoconus or if the right eyehad previously undergone penetrating

keratoplasty), the left eye was used. Thus,quantitative data were obtained from 29eyes. Quantitative analysis was conductedusing dedicated software that came withthe CM (Confocommander 2.7.1, Tomey,Erlangen, Germany). At least three frames

were analysed for each corneal layer andan average was taken. When examiningimages of the endothelium a minimum of 50 cells was evaluated.

During the course of the study, it be-came apparent that the images obtained

of the keratoconic stroma were degradedto varying degrees. Many images were hazy and often had a hyper-reflective appear-ance. Keratocyte nuclei were poorly dif-ferentiated in hazy images, renderinganalysis difficult. To quantify the levels of haze and hyper-reflectivity, a grading scaleof this phenomenon was constructed fromrepresentative images (Figure 2). Themeaning of each level of grading is ex-plained in Table 1. The level of haze ineach 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 sub-sequently available for examination usinglight microscopy (LM). Following surgicalremoval, the corneal buttons were placedin 10 per cent neutral buffered formalin(pH 7.6). The buttons were cut into two

sections along the vertical meridian, dehy-drated through graded alcohols (70, 90and 100 per cent), de-lipidised in xyleneand impregnated with paraffin wax at 56°C.

Patient A had unilateral keratoconus.Limited cross-sections of the central cor-nea of this patient were available and werestained with haemotoxylin and eosin.Serial step sections were prepared fromthe excised cornea of Patient B and werestained in an alternating manner with hae-motoxylin and eosin, Periodic acid-Schiff and Masson’s trichrome.

The histological appearance of the cor-neas of these two patients is consideredbelow in the relevant sections relating toeach corneal substructure.

THE CORNEA IN KERATOCONUS

Superficial epithelial cellsCM images of the superficial epithelialcell layer were obtained in 20 per cent of eyes in the MKS.17–20 A normal ap-pearance5,6 was noted in eight per cent of eyes, all of which were classified as havingmoderate keratoconus. Irregular superfi-cial cells with an elongated or spindle-like

shape were found in 12 per cent of eyes.These patients were classified as havingsevere keratoconus (Figure 3).

The CM appearance of elongated epi-thelial cells has been observed by others.Somodi and colleagues23 reported seeingobviously elongated superficial epithelialcells arranged in a whorl-like fashion.

Wygledowska-Promienska and associates24

noted that desquamating epithelial cells were elongated and arranged aroundthe apex of the cornea. Uçakhan andcolleagues25 observed the epithelial cellsto be elongated in 18 per cent of kerato-conic eyes (all with severe keratoconus).Desquamating, elongated superficial epi-thelial cells were observed in one patient.

Weed and associates26 noted that desqua-mating superficial epithelial cells withbright cell boundaries were easily visiblein keratoconic eyes.

Uçakhan and colleagues25 found that the density of superficial epithelial cells inkeratoconic eyes (942 ± 137 cells/mm2)

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was not significantly different from that of age and gender-matched controls (1,087 ±971 cells/mm2). This contrasts with thespecular microscopy results of Tsubotaand associates,27 who found the mean sizeof superficial epithelial cells to be in-creased in keratoconus.

Mocan and Irkec28 proposed that theinstillation of fluorescein prior to CMcan enhance the imaging characteristicsof this technique. They observed in-creased intracytoplasmic and nuclearstaining of the superficial epitheliumthat was more readily visible after instilla-tion of fluorescein. They suggested that this increased intracytoplasmic and nu-clear staining in the superficial cornealepithelium of patients with keratoconusmight be indicative of increased epithe-lial turnover.

Wing cellsThe wing cell layer of the epitheliumappeared normal5,6 in only eight per cent of eyes, all of which had moderate kerato-conus. In patients with severe keratoco-nus, the wing cell layer displayed large,irregularly spaced nuclei (16 eyes of 12patients) (Figure 4). No images of the

wing cell layer were obtained from theremaining 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 popula-tion (6.4 ± 0.8 µm).

Basal epithelial cellsCM images of the epithelial basal cell layerrevealed considerable inter- and intra-

patient variability. A normal appearanceof visible cell borders with a regulararrangement of cells5,6 was found in eight per cent of eyes, all of which were classi-

fied as having either mild or moderatekeratoconus. The most common finding

was a hazy appearance (22 eyes of 16patients). In 12 per cent of eyes, the basalcell layer had an irregular appearance,

with large cells and faint cell borders(Figure 5). These images showed somesimilarities to the images obtained of the

wing cell layer but were differentiatedfrom wing cell images by virtue of theirlocation adjacent to Bowman’s layer. Most of these eyes were classified as having

severe keratoconus.The average basal epithelial cell diame-

ter in keratoconic patients (11.4 ± 1.2 µm) was significantly greater (p

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were significantly higher than thosereported by Weed and associates26 in bothkeratoconic patients (unpaired t-test:moderate keratoconus t = 3.5, p

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vations in the MKS17–20 confirm these his-tological observations.

Tsubota and associates27 were the first toreport the appearance of elongated super-ficial cells in vivo using the specular micro-

scope. They found elongated superficialepithelial cells, which became sharp andspindle-like in severe cases of keratoconus.Spindle-shaped cells are characteristic of the wound-healing response and its asso-ciated cellular migration34 and the pres-ence of such cells has also been noted inpatients who have undergone penetratingkeratoplasty or epikeratophakia.35 All of the corneas displaying elongated superfi-cial cells in the MKS17–20 were classified assevere keratoconus.

Sub-basal nerve plexusPatel and McGhee36 used laser scanningCM to produce two-dimensional recon-structions of the corneal sub-basal nerveplexus in four eyes of four patients withkeratoconus. This was achieved by havingpatients fixate on targets arranged in agrid to enable imaging of the cornea ina wide range of positions. A mean of 402 ± 57 images was obtained for eachcornea, to create confluent montages(Figure 7). The mean dimensions of the

corneal areas mapped were 6.6 ± 0.7 mmhorizontally and 5.9 ± 0.7 mm vertically.Thus, these authors were essentially ableto elucidate the overall distribution of sub-basal nerves in the living central to midpe-ripheral human cornea in keratoconus.

All corneas exhibited abnormal sub-basal nerve architecture compared withpatterns previously observed in normalcorneas.8 At the apex of the cone, there

was a tortuous network of nerve fibre bun-dles, many of which formed closed loops.

At the topographic base of the cone, nervefibre bundles appeared to follow the con-tour of the base, with many of the bundlesrunning concentrically in this region(Figure 8). Central sub-basal nerve den-sity was significantly lower (p

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None of the keratoconic corneas in thisgroup with a normal appearance of Bow-man’s layer displayed corneal scarring. Alleyes were classified as having moderate orsevere disease.

Bowman’s layer had an abnormalappearance in the remaining 57 per cent of eyes. The type of abnormality variedbetween patients. In many cases, both epi-thelial nuclei and keratocyte nuclei fromthe anterior stroma appeared to be in thesame plane as Bowman’s layer. In someimages of Bowman’s layer, nerve fibresappeared to run in and out of the planeof the field of view. An increased level of haze was apparent in many images, whichcorresponded to increased haze in theanterior stroma. The images of Bowman’s

layer from three patients (three eyes) con-tained hyper-reflective patches. Someimages also displayed what appeared tobe hyper-reflective nuclei of the kerato-cytes (seven eyes). These hyper-reflectivechanges were seen only in patients withapical scarring.

In agreement with the findings of theMKS,17–20 Somodi and colleagues23 and

Wygledowska-Promienska and associates24

noted highly reflective changes near Bow-man’s layer. Somodi and colleagues23 also

observed fold-like structures. These may have been artefacts induced by pressure of the cone tip of the CM against the cornealsurface. Such artefacts have been reportedto occur with the use of CMs that requirecorneal contact 37–39 and the instrument used by Somodi and colleagues23 requiredcorneal contact. Another explanation forthe appearance of fold-like structures isthat these may have been so-called ‘K-structures’, which are features that havebeen reported to appear in the region of Bowman’s layer in the normal cornea.40

Contrary to the above observations, Uça-khan and colleagues25 and Weed andassociates26 were unable to detect any abnormalities in Bowman’s layer in theirkeratoconic patients.

In the MKS,17–20 histological examina-tion of the cornea of Patient B revealedBowman’s layer to progress from a normalsingle layer to an abnormal bilayer, as it approached the apical region and tobecome split and fragmented in the

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 per-

mission from Patel and McGhee.36

A B

400 µm

Figure 8. Electronic tracings of nerve fibre bundles provide schematics devoid of back-

ground data in four keratoconic patients, labelled A, B, C and D. These tracings are

superimposed, to scale, onto the corresponding anterior tangential corneal topographi-

cal maps of these patients. Reproduced with permission from Patel and McGhee.36

BA

C D

Anterior tangentialpower (D)

2 cm

60.00

57.00

54.00

51.00

48.00

45.00

42.00

39.00

36.00

33.00

30.00

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immediate proximity of the apical scar(Figure 9A). The bilayer appearance is evi-dent in the corresponding CM image(Figure 9B), in which keratocyte nucleican be observed to the right of the field

and a generally amorphous field isobserved on the left, with the odd faint keratocyte nucleus and a single nervefibre traversing the frame. The normalCM appearance of Bowman’s layer isshown in Figure 9C. With the CM, Bow-man’s layer appeared as a hyper-reflectivefield (Figure 9D) in the heavily scarredregion of the cone.

Many research groups13,14,16,33,41 havereported the presence of breaks and dis-continuities in Bowman’s layer in kerato-conus using LM and both transmission

and scanning electron microscopy. Sawa-guchi and colleagues16 used scanningelectron microscopy to examine the kera-toconic cornea and found breaks in Bow-man’s layer and irregular thinning. Thesefindings are consistent with the appear-ance of this layer with the CM in themajority of the keratoconic eyes in theMKS.17–20 LM of the cornea of Patient Bconfirmed that the irregular appearanceof Bowman’s layer using CM was consis-tent with fragmentation and breaks in

Bowman’s layer.The ruptured areas of Bowman’s layer

have been reported to be filled with eitherepithelium or proliferated collagenous tis-sue that is derived from the anteriorstroma.13,14,16,33,41 Chi, Katzin and Teng33

documented keratoblasts and newly formed connective tissue in areas whereBowman’s layer had been destroyed. Inthe MKS,17–20 LM examination of the cor-nea confirmed that hyper-reflective nucleiseen with the CM corresponded to fibro-blastic cells. Histological examination of the cornea of Patient B demonstrated con-siderable disruption to Bowman’s layer inthe region of the apical scar, which con-sisted of abnormal collagenous materialand fibroblasts. These findings are inagreement with the observations of Chi,Katzin and Teng.33 These authors de-scribed the presence of keratoblasts, how-ever, at the time that their study wasconducted, it was not known that kerato-cytes were able to be activated into a fibro-

blastic status. This was demonstrated many years later.42

Stroma Stromal images of the central corneaobtained by CM showed varying amountsof haze and hyper-reflectivity. Extremelevels of haze were present in 44 percent of eyes. When visible in thesecorneas, the keratocyte nuclei oftendisplayed an irregular, hyper-reflectiveappearance. Severe haze was found tocorrespond with apical scarring on SLBevaluation in 35 per cent of eyes. Thefour eyes in which apical scarring wasnot apparent when viewed with the SLBdisplayed less severe levels of haze onCM. The remaining eyes showed only mild degrees of haze. In these patients,keratocyte nuclei were easily distin-guished and had an appearance similarto that seen in the normal eye.5,6

The level of haze was quantified usingthe grading scale (Figure 2). The pres-ence of scarring on SLB examination wassignificantly related to the level of hazeseen on CM. This was true for both theanterior (F = 7.6, p

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(r2 = 0.16, F = 4.6, p = 0.04). These find-ings support the validity of the kerato-conic haze grading scale. Surprisingly, thedegree of stromal haze was not shown tobear any relationship to disease severity as

classified by corneal curvature.Haze in the corneal stroma of kerato-conic eyes, especially the anterior stroma,has also been noted by others, in agree-ment with the MKS.17–20 Uçakhan andcolleagues25 reported increased back-ground illumination and reflectivity, andirregular arrangement of stromal kerato-cyte nuclei in the anterior stroma of 29per cent of eyes. They suggested that thisappearance was consistent with varyingdegrees of haze and stromal scarringobserved using SLB.

Wygledowska-Promienska and associ-ates24 noted an apparent disarrangement of collagen fibres reflected by bright background illumination in the anteriorregion of the stroma beneath Bowman’slayer. Somodi and colleagues23 alsoobserved increased reflectivity in the ante-rior stroma. In the posterior stroma, kera-tocytes had extremely long almost parallelprocesses, however, in scarred stroma, thekeratocytes were spindle-shaped andarranged irregularly.

Keratocyte density An assessment of stromal KD in keratoco-nus is confounded by two key factors. First,patients with keratoconus are typically fit-ted with rigid contact lenses to neutralisecorneal distortion and afford satisfactory

vision. The more severe the condition, themore likely it is that rigid lenses are being

worn. With the exception of one researchgroup,43 the general consensus in the lit-erature is that, in normal subjects, contact lens wear causes an apparent reduction inKD.44–48 This is thought to occur as a result of the physical impact of lenses on thecorneal epithelium, which releases inflam-matory mediators that cause keratocyteapoptosis.49 Thus, there is a need to deter-mine whether the reduction in KD associ-ated with keratoconus is due to the effectsof lens wear or the direct pathologicaleffects of keratoconus or possibly both.Second, as discussed above, the cornealstroma in keratoconus is often hazy and it

is difficult to see keratocytes in the pres-ence of significant haze, leading to apotential under-estimation of KD in suchcases.

The four studies19,25,26,44 that haveaddressed the question of KD in keratoco-nus adopted different approaches inattempting to account for these con-founding influences. Table 2 provides a

summary of estimates of KD published inthese works.19,25,26,44 In reviewing the datain this table, it should be noted that theabsolute cell densities reported by Erieand associates44 can not be directly com-pared with the other data displayed inthe table because Erie and associates44

expressed cell density as a volumetricmeasure (cells/mm3), whereas all otherdata are expressed as a function of cellarea (cells/mm2). It is not possible to con-

vert between the two units because thedepth of the CM sections used to calculatefield volume was not stated by Erie andassociates.44

Most of the keratoconic patients in theMKS17–20 were wearing rigid contact lenses,so a control experiment 50 was conductedto determine the effects of rigid lens wearon KD in non-keratoconic subjects. Slit scanning CM (Tomey Confoscan P4) wasused to evaluate KD in 22 subjects whohad been wearing rigid lenses on a long-term, daily wear basis. These data were

compared to those of 22 non-lens-wearingcontrol subjects. Subjects with a pre-

vious history of polymethyl methacrylate(PMMA) lens wear showed a reduction(p

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P = 0.005) but not PKD. Post hoc analysisrevealed that atopy (p = 0.009), a history of eye rubbing (p = 0.006) and cornealstaining (p = 0.035) were each signifi-cantly associated with AKD. Contact lens

wear, race and the presence of a scar wereunrelated to AKD or PKD.

Erie and associates44

measured KD infour groups: lens-wearing and non-lens-wearing keratoconic patients andlens-wearing and non-lens-wearing non-keratoconic control subjects. Amongthose who did not wear contact lenses, nodifference in AKD and PKD was foundbetween keratoconic patients and non-keratoconic control subjects. Among con-tact lens wearers, AKD and PKD werefound 31 and 41 per cent lower in kerato-conic patients compared with non-keratoconic control subjects, respectively.These authors concluded that KD is nor-mal in keratoconic patients but keratocyteloss is somehow exacerbated by lens wear.

As Erie and associates44 excluded patients with severe keratoconus from their study,interpretation of their results must beconfined to changes that occur in mild tomoderate keratoconus.

In the study of Uçakhan and col-leagues,25 only non-lens wearing kerato-conic and control subjects were

examined. These authors found AKD andPKD to be 19 and 22 per cent lower thanin controls, respectively. Although thisfinding contradicts that of Erie and asso-ciates,38 who found no difference in AKDor PKD between non-lens-wearing kerato-conic and control subjects, it should be

noted that the keratoconic patients in theexperiment of Uçakhan and colleagues25

were not confined to those with mild tomoderate disease. In fact, 54 per cent of their experimental group were classifiedas having severe keratoconus. Uçakhanand colleagues25 did not explain how their patients with severe keratoconusmanaged to see, given that they appar-ently were not corrected with rigid con-tact lenses or any other form of contact lenses. Considered together, the findingsof Erie and associates38 and Uçakhan andcolleagues25 suggest that keratocyte loss inkeratoconus may be related to diseaseseverity.

Weed and associates26 assessed patients with moderate and advanced keratoconus who wore contact lenses and found signif-icantly higher KD compared with non-lens-wearing control subjects. Specifically,

AKD was 14 per cent higher in patientsdisplaying moderate keratoconus, and

AKD and PKD were 20 and 16 per cent

higher in patients with advanced keratoco-nus. Compared with lens-wearing controlsubjects, AKD and PKD were 31 and 15per cent higher in patients displayingmoderate keratoconus, and 36 and 22 percent higher in patients with advancedkeratoconus. The findings of Weed and

associates26

of higher KD in keratoconicpatients directly contradict those of theMKS17–20 of a lower KD in keratoconicpatients.

In view of the different approaches out-lined above in determining KD in kerato-conic patients, it is difficult to reconcilethe results of these works. Three of thefour papers that addressed this issue19,25,44

indicate a lower KD in keratoconus but the extent to which these changes reflect the effects of lens wear versus the under-lying pathological changes in keratoconusremains unclear. One possibility, sug-gested by Kallinikos and Efron49 is that changes in KD may be a function of lateralcell migration and redistribution as wellas, or instead of, cellular apoptosis. A combination of these phenomena couldexplain both increases and decreases inKD.

The original idea—conceived half acentury ago by Chi, Katzin and Teng13,33—that the earliest ultrastructural changes in

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

Author Year Anterior stroma Posterior stromaKeratoconus Control p-value Keratoconus Control p-value

Erie and colleagues44 2002 24,564 ± 8,750b 35,630 ± 3,858b p

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keratoconus occur at the epithelial base-ment membrane, has been extendedrecently by Wilson and colleagues,45 whosuggest that epithelial damage causes a

reduction in AKD through apoptosis. Thisis thought to be triggered by cytokines(including interleukin 1 and Fas-ligand)released from the damaged epithelialcells.51 An increased number of anteriorkeratocytes exhibit signs of apoptosis inthe keratoconic cornea when compared tonormal corneas and corneas sufferingfrom other diseases.52 It has also beenshown that the keratocytes within the kera-toconic cornea have four times as many receptors for interleukin 1, potentially sensitising them to this cytokine.53 CMreports of reduced AKD in kerato-conus19,25,44 and the LM observationsfrom the MKS17–20 are consistent with thenotion that keratocyte apoptosis inducedby epithelial damage is one of the mecha-nisms responsible for the reduction in

AKD in keratoconus (Figure 11). The clin-ical evidence of this is the presence of sig-nificant corneal fluorescein staining inkeratoconic patients as observed in theMKS.17–20

Confocal versus light microscopy In the MKS,17–20 hyper-reflective keratocytenuclei and stromal haze were apparent

when examining CM images of the cornea

of Patient B (Figure 12). Evaluation of theserial step sections prepared for LMrevealed the presence of disorganised tis-sue, confirming the SLB appearance of apical scarring in this patient. The scarredregion measured approximately 220 µm at its widest point. Accurate measurement of hyper-reflective regions in CM images wasnot possible as there was no defined bor-der, however, the size of the regions of hyper-reflectivity observed with the CM

was roughly consistent with measurements

of the scar taken from the histologicalsamples.Examination of tissue sections from the

cornea of Patient B at higher magnifica-tion revealed a dense accumulation of fibroblasts in the region of the scar. Nuclei

were rounded and more irregular inshape compared to the elongated, flat-tened appearance of normal keratocytenuclei.5,6 The extra-cellular matrix washighly irregular compared to the non-scarred peripheral area of the same cor-

nea. Some of the images obtained fromCM of Patient B contained a mixture of hyper-reflectivity and evidence of epithe-lial nuclei and are thought to represent

images taken from near the apex of thescar.

Previous ultrastructural studies haveshown the keratoconic stroma to be dis-torted in regions where there are breaksin Bowman’s layer.16,35,54 Fibrillar degener-ation and fibroblastic accumulation havebeen demonstrated in the stroma beneaththese breaks.33 Keratocyte morphology hasalso been shown to be abnormal in thekeratoconic eye.13 These observations areconsistent with CM observations in theMKS17–20 of significant abnormalities of keratocyte nuclei, stromal haze andhyper-reflectivity. Hyper-reflective kerato-cyte nuclei probably represent fibroblastsas observed with the LM.

Research into the wound-healingresponse of the stroma has revealed thepresence of hyper-reflective keratocytenuclei. These have been referred to asactivated keratocytes, that is, keratocytesactivated to a repair phenotype (or fibro-blasts). Using rabbit corneas, Møller-

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

Normal cornea Keratoconus

Interleukin-1

Epithelial trauma

EndotheliumA B C D

Stroma

Epithelium

Keratocyte

Receptor forInterleukin-1

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Pedersen and co-workers55 showed that thehaze apparent on CM following photo-refractive keratectomy was due to theincreased reflectivity of migrating and acti-

vated keratocytes. Similar findings havebeen documented in humans. In the early stages of keratocyte activation, the nucleibecome more apparent. A more extreme

wound-healing response results in the cellbodies becoming visible.56 Transmissionelectron microscopy has shown that thesehyper-reflective cells represent keratocytesactivated to a repair phenotype.57 TheMKS17–20 demonstrated what appears to beactivation of keratocytes in association

with apical scarring in the keratoconic eye.The apparent association between stromalhaze and the appearance of activated kera-tocytes suggests that keratocyte hyper-reflectivity may serve as a useful marker of disease progression in longitudinal studiesof keratoconus, which could be monitoredin vivo using CM.

The extreme levels of stromal hazeobserved with the CM cannot be attrib-uted solely to an accumulation of fibro-blasts. Keratocytes are known to beresponsible for the production of theextracellular matrix and in turn, thisaffects the arrangement of collagen withinthe corneal stroma. LM of the cornea of Patient B revealed the presence of abnor-mal collagenous tissue surrounding fibro-blasts. Studies using X-ray diffraction haveshown that the normal arrangement of collagen fibres is severely disrupted inscarred regions of the keratoconic eye.58

This is clearly demonstrated by the histo-logical investigations performed in theMKS.17–20 The irregular arrangement of this tissue will contribute significantly tothe resulting stromal haze observed usingCM as the normal lattice arrangement of the collagen fibres is disrupted. Regulararrangement of the collagen fibres

within the corneal stroma is known to be

partly responsible for the transparency of the cornea.59 Møller-Pedersen and collea-gues55 suggested that the deposition of anew extra-cellular matrix may also contrib-ute to corneal haze following photorefrac-

tive keratectomy.

Stromal nervesSimo Mannion, Tromans and O’Donnell60

investigated stromal nerve morphology and corneal sensitivity in 13 patients withkeratoconus and 13 age-matched controlsubjects, using in vivo CM and non-contact (‘air puff’) corneal aesthesiome-try. Stromal nerve fibre density was foundto be significantly lower in keratoconicpatients (1,018 ± 490 µm) versus controlsubjects (1,821 ± 790 µm) (p = 0.006). The

mean diameter of stromal nerve fibres was found to be greater in patients withkeratoconus (10.2 ± 4.6 µm) compared tocontrol subjects (5.5 ± 1.9 µm) (p = 0.007).The orientation of stromal nerve fibres inthe patients with keratoconus appearedto be altered from the predominantly

vertical orientation seen in controlsubjects.

Corneal touch threshold was similar inthe two groups, although corneal sensitiv-ity in patients with keratoconus using con-

tact lens corrections (1.18 ± 0.19 g/mm2

) was reduced (that is, the ‘air puff’ pres-sure needed to be higher to elicit asensation) compared to the contact lens-

wearing control subjects (0.98 ± 0.05 g/mm2) (p = 0.03). Simmo Mannion andcolleagues60 concluded that there is asignificant reduction in stromal nervedensity in the keratoconic cornea, thereduced stromal nerve density is a causeof the reduced corneal sensitivity in kera-toconic contact lens wearers and the thick-ened stromal nerve fibres observed inkeratoconic corneas may explain why prominent stromal nerves are often seenusing SLB in such patients.61

Corneal nerves may play an active rolein the degenerative changes that occur inkeratoconus, by facilitating keratocyte-epithelial interactions. Brookes andassociates62 observed nuclei of aberrant anterior keratocytes wrapping aroundnerves as they passed through the other-

wise acellular Bowman’s layer from the

Figure 12

A. CM image of hyper-reflective and distorted keratocyte nuclei in Patient B, possibly

representing activated fibroblasts.

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

nuclei. Normal keratocytes are present below this region ( ¥ 40 objective).

Keratoconus

Normal cornea ‘Normal’ keratocyte nuclei

A

B

C

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stroma to the epithelium. As the keratoco-nus progressed and Bowman’s layerdegraded, these keratocytes were seen toexpress higher levels of the lysosomalenzymes cathepsin B and G and to

become displaced anteriorly into the epi-thelium. Localised nerve thickening alsodeveloped within the epithelium in associ-ation with cathepsin B and G expressionand appeared to be destructive to the cor-nea. Specifically, the authors noted that enzyme activity by keratocytes seemed tobe causing localised structural degrada-tion of the anterior stroma, leading tonear-complete destruction of both Bow-man’s layer and the stroma. Observationsof apparent intermixing of epithelial cells,keratocytes from the anterior stroma and

nerve fibres within split sections of Bow-man’s layer made in the MKS17–20 usingCM and LM, support the keratocyte-epithelial interaction theory of Brookesand associates.62

Striae Alternating dark and light bands wereobserved with CM in the stromal imagesof 45 per cent of keratoconic eyes exam-ined in the MKS.17–20 The bands corre-sponded with the appearance of Vogt’s

striae on SLB examination. Figure 13A shows a SLB image of striae visiblein a keratoconic patient. When magni-fied, the image of the striae taken withthe SLB (Figure 13B) is strikingly similarto the CM image of bands in the pos-terior stroma of a keratoconic patient (Figure 13C).

Bands observed with the CM were most commonly in the posterior stroma. Poste-rior bands varied in width, ran mainly ina near vertical direction and appeared torun a straight course through individualimage frames. Keratocyte nuclei werelocated in between the bands but theirdistribution appeared unaffected by thepresence of bands. Nerve fibres appearedto run a straight course through thebands. When present, bands in the ante-rior stroma showed greater variability in

width and direction within a singleframe. Bands were present only in theanterior stroma in more severe levels of keratoconus. No obvious correlate of

banding could be observed with the LM(Figure 14).

Uçakhan and colleagues25

observedfolds, which they referred to as Vogt’sstriae, in 50 per cent of the keratoconiceyes they examined. Folds were seen in theanterior 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 (50per cent). Interestingly, their descriptionof stromal folds as representing ‘crests andtroughs’ suggests that they believe folds tohave a three-dimensional construct. Forexample, Uçakhan and colleagues25 statedthat keratocyte nuclei were visible only over the light bands, which they called‘crests’, and were not seen on longitudinaldark bands, which they believed corre-sponded to ‘troughs’. Posterior stromalfolds were observed in 14 eyes with severekeratoconus, eight eyes with moderatekeratoconus and two eyes with mild kera-toconus. In the earlier studies of Wygle-dowska-Promienska and associates24 andSomodi and colleagues,23 folds wereobserved only in the posterior stroma.

The images obtained in the MKS17–20

suggest that the stromal bands seen

with the CM represent collagen lamellaeunder stress rather than folds. Komaiand Ushiki63 demonstrated a differentialarrangement of collagen lamellae in theanterior and posterior corneal stroma.

Anterior lamellae are 0.5 to 30 µm wide,they have a flat tape-like shape, run inrandom directions and are often inter-twined. The lamellae of the posteriorstroma are wider (100 to 200 µm) andhave the appearance of broad sheets.63

The CM images obtained of bands inthe anterior and posterior stroma show a similar pattern to those describedby Komai and Ushiki.63 Anterior stromalbands are narrower and irregularly spaced.

A tape-like shape was observed in somepatients. The pattern of banding varied insequential frames. Bands in the posteriorstroma were wider, regularly spaced andoften consistent in direction in severalsequential frames. If clinically observed

Vogt’s striae represented folds (ratherthan lines of stress), then their appearance

Figure 13

A. SLB image of a keratoconic cornea, 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

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on CM would not be expected to be

related to the pattern of collagen lamellae.The transparency of the corneal stroma

in the normal eye is, in part, due to theregular and precise arrangement of thecollagen fibrils. Maurice59 demonstratedthat for the corneal stroma to be transpar-ent, it is necessary that fibrils are parallel,equal in diameter and have their axes dis-posed in a regular lattice formation. Thisregular arrangement results in mutualinterference of the light rays leading tominimal light scattering.59 This effect islikely to be greatest at the posteriorstroma, as a result of the more regularfibril arrangement found posteriorly.64

The banded appearance of the stromaobserved on CM in keratoconic patientsmay represent a widespread, irregularseparation of individual collagen fibrils

within the lamellae. Indeed, a numberof authors65–67 has demonstrated markedabnormalities in the organisation of theanterior corneal collagen lamellae of keratoconic corneas.

Disruption to the arrangement of col-

lagen lamellae at any level of the stroma will cause light to be refracted differently,thereby having an effect on the mutualinterference of light rays passing throughthe corneal stroma. The CM images con-taining stromal bands demonstrate varia-tions in contrast, presumably due to thedisrupted arrangement and irregular sep-aration of the collagen fibrils. CM imagesof stromal bands in keratoconus do not have the uniformity of images obtained of collagen fibrils in the normal cornea.58,65

Both of these findings indicate significant alterations of the collagen fibre arrange-ment in the keratoconic eye. This may bepartly responsible for the reduced visionin keratoconic patients.

Tripathi and Bron68 described theappearance of a secondary mosaic in thecornea, the structural basis of which lies inthe particular arrangement of many prom-inent collagen lamellae of the anteriorstroma that take an oblique course to gaininsertion into Bowman’s layer. This can

not be seen in the normal eye becauseBowman’s layer is under tension due tointraocular pressure. However, Dangeland Kracher69 observed the mosaic pat-tern in 75 per cent of eyes of keratoconic

patients wearing rigid lenses versus fiveper cent of non-keratoconic rigid lens wearers. These observations introduce thepossibility that the disarrangement of thecollagen network in the keratoconic eyesomehow facilitates the appearance of amosaic pattern when the cornea is stressedby the pressure of a rigid lens. Thus, theappearance of dark bands in keratoconicpatients in the MKS (50 per cent of whom

were wearing rigid lenses) may be, at least in part, a manifestation of this mosaicformation.

The direction of the bands in the poste-rior stroma was found to correlate well

with the steepest Sim-K axis of the corneaas determined by corneal topography.18

Therefore, it seems probable that the ori-entation of the bands is due to a patternof stress in the collagen lamellae emergingfrom the apex of the cone. By way of exam-ple, consider Figure 15A, which is a sche-matic representation of a presumed ‘stresspattern’, templated on top of a topograph-ical map of a keratoconic cornea, which

may emanate from the apex of an infero-nasally located cone.

If the objective lens of the CM were tobe positioned for examination of the cen-tral cornea, an area superior and slightly temporal to the cone apex would be exam-ined (Figure 15A, red box). Stress lines

would run through the field of view at about 80 degrees (using conventionalophthalmic lens axis notation). This

would result in dark banding as shown inFigure 15B. If the objective lens of the CM

were to be positioned for examination of the apex of the cone (Figure 15A, bluebox), stress lines might be seen runningthrough the field of view at a variety of angles. This would result in dark bandingas shown in Figure 15C.

The orientation of banding in the stro-mal images of keratoconic patients ob-served in CM images captured from thecentral cornea are consistent with thisschematic model. In many patients, thecones were located inferiorly to the cen-

Figure 14. Bands observed with CM in keratoconus

A. Bands of varying width in the anterior stroma, running orthogonally at approximately

90 degrees and 180 degreesB. Fine vertical bands in the anterior stroma. A bifurcating nerve fibre and keratocytes

are visible.

C. Vertically oriented bands in the mid-stroma

D. Vertically oriented bands in the mid-stroma with faint horizontal banding

E. Bands in the posterior stroma, with a nerve fibre crossing horizontally. Keratocytes

are visible only between the dark bands.

F. Widely-spaced dark bands in the posterior stroma

Anterior stroma Mid stroma Posterior stroma

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tre of the cornea and the banding wasnear-vertical. The findings for the pa-tient with a centrally located cone are alsoconsistent with this theory. This patient displayed highly irregular posterior band-ing, namely, faint bands were apparent horizontally in addition to more promi-nent vertically orientated bands. Thismixed banding pattern indicates that thestress in the cornea corresponds to theapex of the centrally located cone. Fur-ther research would need to be under-taken, by way of imaging bandingpatterns at various locations on kerato-conic corneas, to test the hypothesis that banding represents stress lines emanatingfrom the cone apex.

X-ray scattering has unambiguously demonstrated that the majority of col-lagen fibrils in the central cornea adopt apreferred orientation in the inferior-superior and nasal-temporal directions.70

If observations of banding (with the CM)and striae (with the SLB) in the central

cornea are related to the orientation of collagen fibrils, then these formations

would be expected to be found horizon-tally as well as vertically. On examination

with CM in the MKS,17–20 horizontal and vertical bands were observed in the ante-rior stroma and predominantly verticalstriae were observed in the posteriorstroma. Vogt’s striae in the same patientsseemed to be predominantly oriented

vertically when viewed with the SLB. Although the early literature71 suggeststhat Vogt’s striae are primarily vertically oriented when observed with the SLB,more recent anecdotal SLB observationsof striae in keratoconus patients (GavinO’Callaghan, personal communication)indicate that striae can occur at any orien-tation (Figure 16). In addition, based ontheir observations of photographic imagesof striae captured from over 1,500 kerato-conic patients, senior authors of the CLEK study 21 are of the opinion that striae inkeratoconus can occur at any angle (Karla

Zadnik, Joe Barr and Timothy Edrington,personal communication).

Smolek and McCarey 72,73 studied thecohesive strength of corneal lamellaeacross the cornea. Investigations of the

lamellae in the vertical meridian haveshown that the inferior cornea has theleast cohesive strength.73 Varying patternsof cohesive strength were seen betweenindividuals but paired corneas often dis-play the same strength profiles. Smolek73

reported a circumstantial correlationbetween cohesive strength and the pat-terns seen in the different forms of cor-neal ectasia.

In keratoconus, the cone is most oftenlocated inferiorly or centrally,74 corre-sponding to the areas of reduced strength

found by Smolek.73 The appearance of stromal banding in the MKS17–20 may rep-resent a stress-related change in corneallamellae, corresponding to areas of reduced strength in the cornea and theformation of an ectatic cone-like protru-sion in that area. Examination of otherectatic degenerations such as keratoglo-bus and pellucid marginal degenerationmay shed further light on this hypothesisby revealing different patterns of bandingcorresponding to regions of reduced

cohesive strength of stromal lamellae andclinical evidence of ectasia.

Desçemet’s membraneNo abnormalities were detected with theCM at the level of Desçemet’s membranein the MKS;17–20 however, Wygledowska-Promienska and associates24 observed cen-tral detachment of the Desçemet’s mem-brane and the endothelium from thestroma in advanced keratoconus. Uça-khan and colleagues25 observed folds at the level of Desçemet’s membrane in eight per cent of keratoconic eyes.

Using LM, Chi, Katzin and Teng33

observed folds and buckling at the level of Desçemet’s membrane in the later stagesof keratoconus and ruptures were ob-served in Desçemet’s membrane in 12per cent of corneas. These defects werefilled first with endothelial cells and later

with a newly formed membrane. Rupturesin Desçemet’s membrane are thought tobe associated with previous cases of cor-

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

A B

C

Nasal Temporal

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neal hydrops. That Desçemet’s mem-brane was normal in the MKS17–20 is not surprising in view of the absence of a pre-

vious history of hydrops in any of thepatients.

EndotheliumThe endothelial images obtained fromone patient in the MKS17–20 displayed evi-dence of elongated cells (Figure 17). Thisappearance was verified by a masked inde-

pendent observer who was experienced inevaluating CM images. The region of thestroma anterior to these elongated cellsand the remainder of the field of theendothelium adjacent to the elongatedcells appeared normal. There was no evi-dence of elongated endothelial cells inany 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 todisplay a large number of smaller cells

with only scattered large cells, however,there was no difference in endothelialpolymegethism between keratoconic pa-

tients (0.35 ± 0.05) and control subjects(0.38 ± 0.07) (paired t-test: t = 1.8, p =0.08).

Pleomorphism and enlarged endothe-lial cells were seen in 13 per cent of eyes

with severe keratoconus by Uçakhan andcolleagues.25 In one (two per cent) eye

with severe keratoconus with no identifi-able history of acute hydrops, folds inDesçemet’s membrane and endothelialguttata were observed. These authorsfound no difference in mean ECD ormean endothelial cell area between kera-

toconic patients and controls. In eyes with severe keratoconus, the mean ECD was lower than in eyes with moderate(p

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technique, Esgin and Erda79 demon-strated an increase in central ECD follow-ing wear of high oxygen transmissiblerigid lenses. In the majority of cases, myo-pia and rigid lens-wear are features of

keratoconus. This may account for theincreased 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 wasnot possible to correlate these findingsagainst those from CM as the endotheliumof Patient B was obscured by high levels of haze in the anterior cornea.

In the early stages of keratoconus, theendothelium has a normal appearance

when viewed with the LM.33 In moreadvanced cases, it shows flattening and

the nuclei are further apart.33 Specularmicroscopy has revealed an increase inpleomorphism and also a high proportionof 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 coneapex.75 Such observations are consistent

with the notion that corneal tissue is being‘stretched’ as a result of ectasia. In theMKS,17–20 evidence of endothelial cellelongation was observed in only one

patient. The lack of cellular elongation inthe majority of the study group may beattributed to the fact that the central cor-nea (thus typically not the centre of thecone) was imaged in all patients.

LM of the endothelium of Patients A and B showed the cells of this layer to benormal in appearance. Endothelial celldegeneration has been reported in cor-neas with more severe levels of keratoco-nus, with the damage being more preva-lent 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 cen-tral cornea was investigated and the endot-helium beneath the cone was often ob-scured by haze and scarring.

KERATOCONUS AND CONCURRENT

CORNEAL DISEASE

The CM has been used to examine casesof disease that have occurred in the cor-

nea of keratoconic patients. Such stud-ies are important because they canprovide unique insights into the kerato-conic cornea by revealing how this tissueresponds to the stress of additionalpathology.

Acute hydropsGrupcheva and associates80 reported thecase of a Caucasian man with a history of keratoconus since teenage years. He pre-sented with unusual bilateral keratoconus

with acute hydrops that had developed

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

authors

Author Year Keratoconus Control p-value

Hollingsworth, Efron and Tullo19 2005 3,250 ± 352b 3,056 ± 365c p 0.05)

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 indicatenuclei of endothelial cells.

Endothelial cell nuclei

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over a three- to four-month period. Thepatient 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 defor-mation of each cornea was evident withthe SLB, with well-circumscribed oedemaat the apex of the cone, which was slightly more prominent in the cornea of the right eye. No other pathology was highlightedon clinical examination. Orbscan topogra-phy could not be performed as a result of decreased corneal transparency. Accurateintraocular pressure measurement was not possible.

Anterior stromal oedema was observed, with marked subepithelial bullae and folds

in Desçemet’s membrane. Corneal scar-ring was present only anteriorly at thelevel of Bowman’s layer. These in vivo CMfindings confirmed the diagnosis of kera-toconus with a less common bilateral pre-sentation of acute hydrops.

Epidemic keratoconjunctivitis Alsuhaibani, Sutphin and Wagoner81

reported the case of a 14-year-old Saudigirl with keratoconus who developedsub-epithelial infiltrates after the onset

of bilateral epidemic keratoconjunctivitis.CM of the left cornea, conducted eight

weeks after the onset of the infection,showed many highly reflective dendriticcells at the level of the basal epitheliumand anterior stroma. Many highly re-flective fusiform and round cells wereobserved within the anterior stroma, withdecreasing density in progressively deeperlayers of the stroma. These findings werenot present on CM that had been per-formed two weeks before the onset of epidemic keratoconjunctivitis. In this case,CM examination provided clear evidenceof an inflammatory response localised tothe basal epithelium and anterior stromaof the central cornea.

MEDICAL AND SURGICAL

INTERVENTIONS IN KERATOCONUS

A number of medical and surgical ap-proaches can be applied to the treat-ment of keratoconus. The CM facilitates

investigation of the efficacy of these treat-ments in uncomplicated cases and allowsadverse reactions to be studied at a cellu-lar level.

Riboflavin-UVA-induced collagencross-linking Wollensak, Spoerl and Seiler82 havedescribed the technique of riboflavin/ultraviolet A (UVA)-induced collagencross-linking, which is designed to bringthe progression of keratoconus to ahalt. The underlying theory is that there will be an increase in corneal bio-mechanical stiffness due to enhancedcollagen crosslinking as a result of thetreatment.

Mazzotta and colleagues83 assessed cor-

neal tissue modifications using this treat-ment in a group of 10 patients withprogressive keratoconus, as well as regen-eration of the epithelium and subepithe-lial nerve plexus, using the HRT II CM.Treatment included instillation of a 0.1%riboflavin/20% dextran solution five min-utes before UVA irradiation and every fiveminutes for a total of 30 minutes thereaf-ter. A dual UVA (370 nm) light-emittingdiode was used to generate radiant energy of 5.4 Joule/cm2. The protocol included

the operation followed by antibiotic med-ication and eye dressing with a soft thera-peutic contact lens.

After five days of soft contact lens wear,the corneal epithelium displayed a regu-lar morphology and density with CM. Dis-appearance of subepithelial stromalnerve fibres was observed in the centralirradiated area where initial reinnerva-tion was observed microscopically onemonth after the operation. No changes innerve fibres were observed in the periph-eral untreated cornea, with a clear lateraltransition between the two areas. Sixmonths after the operation, the anteriorsubepithelial stroma was recolonised by nerve fibres with restoration of cornealsensitivity.83

A similar pattern of disappearance andregeneration of keratocytes was observedusing CM.84 A reduction in KD in the ante-rior and intermediate stroma and stromaloedema, was observed immediately aftertreatment. Keratocytes were observed to

repopulate the central cornea threemonths after the operation and theoedema had disappeared. At six monthspost-operatively, keratocyte repopulation

was complete. No endothelial damage was

observed at any time.

Intrastromal corneal ring implantsIntrastromal corneal ring implants arecorneal inlays made of plastic, with an arclength of 150 degrees, which are used forthe correction of low to moderate myopia.The outward radial tension of these ringsleads to a reduction in curvature of thecornea and a normalisation of cornealtopography, resulting in reduced myopia,less optical aberration and improved

vision. More recent developments of this

technique include short arc length seg-ments (130 degrees) for the correction of myopia concurrent with astigmatism andradially-placed corneal inlays for the cor-rection of hyperopia.

Ruckhofer and colleagues85 used CMto examine the corneas of a series of keratoconic patients who had implantsinserted 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 intrac-ellular lipids.

Kymionis and associates86 examined 17eyes of 15 patients with keratoconus aged24 to 52 years (mean: 34 ± 11 years), whohad completed five years after insertion of two intrastromal segments of 0.45 mmthickness in the cornea of each eye. Whenexamined using CM, most patients exhib-ited normal central corneal morphology in all layers, with normal epithelial cells,subepithelial nerve plexus, keratocyte dis-tribution and endothelial morphology.Needle-shaped keratocytes and tortuoussub-basal nerves were observed within thestroma in one patient. Microdeposits,stretched keratocytes and mild fibrosis

were observed at or close to the anteriorchannel of all patients. At the plane of the implant, oval-shaped deposits wereobserved along the channel. One patient exhibited increased fibrosis or collagendisruption a few microns away from thering segment.

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Epikeratophakia Shi and colleagues87 used CM to study 24 cases of keratoconus from three daysto five years after epikeratophakia. Thetissue lens was observed to be covered

by apparently ‘flattened’ superficial cor-neal epithelial cells three to four dayspost-operatively. Epithelial wing andbasal cells were also observed but themorphology and arrangement of thesecells were irregular with low cell density.The superficial flat epithelial cells ap-peared normal at one month and themorphology and density of the basal epi-thelial cells tended to be normal at sixmonths post-operatively. The subepithe-lial nerve plexus appeared irregular at 18 months but appeared normal two

years post-surgery.In the stroma of the tissue lens, kerato-

cytes appeared circular, dot-shaped, rod-shaped or reticular. A few normal kerato-cytes were observed at the periphery of thetissue lens two years post-operatively. At five years, KD in the periphery remainedlower than that in the centre of the tissuelens. Stromal nerves appeared in the tissuelens six months after surgery and the quan-tity of nerves had increased after two yearsbut was still less than normal after five

years. There was no change in the stromaand endothelium of the recipient cornea.

Penetrating keratoplasty A longitudinal evaluation of four patients who had undergone penetrating kerato-plasty was undertaken by Hollingsworth,Efron and Tullo20 for 12 months aftersurgery, using slit scanning CM. The pro-cedure was preformed because of kerato-conus (two patients), Fuch’s dystrophy and lattice dystrophy.

Patients were examined on four occa-sions over a 12-month period after sur-gery. The epithelium varied in appearancebetween patients and took at least 12months to appear normal. Bowman’s layer

was viewed as an acellular layer immedi-ately after surgery with no evidence of nerve fibres, although some nerve compo-nents were apparent 12 months aftersurgery. Stromal nerves were not visibleimmediately after surgery. One year fol-lowing penetrating keratoplasty, there was

evidence of thin nerves running a straight course through the central stroma. AKDand PKD were lower in the transplantedcornea and appeared to remain constant over a period of 12 months.

Activated keratocytes were seen in theanterior stroma of all patients. They appeared to be responsible for signifi-cant levels of corneal haze. The time

within which this keratocyte activation oc-curred varied between individuals. ECDdecreased at an accelerated rate over the12-month period.

Imre, Resch and Nagymihaly 88 exam-ined seven eyes with clear grafts at 15 and66 months after penetrating keratoplasty.The preoperative diagnoses were kerato-conus (two), granular corneal dystrophy

(two), pseudophakic bullous keratopathy (two) and corneal ulcer (one). Mean den-sity of basal epithelial cells was 3,928 ± 378cells/mm2 at 15 months and 3,284 ± 565cells/mm2 at 66 months post-operatively.

At 15 months, AKD and PKD were 750± 113 and 601 ± 98 cells/mm2, respec-tively, 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 ± 272cells/mm2 at 66 months. The results of

this study are consistent with the findingsof the MKS.17–20 Both were longitudinalevaluations and suggest that there is anongoing decline in the cellular integrity of corneal grafts up to six years followingpenetrating keratoplasty.

Niederer and colleagues89 conducted across-sectional CM study comparing cor-neas from 42 patients after penetratingkeratoplasty with those of 30 controls.Patients were assessed by ophthalmichistory, clinical examination and comput-erised corneal topography. Time after sur-gery ranged from one month to 40 years(mean: 85 ± 105 months). Significant reductions in epithelial CD (p

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© 2007 The Authors


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