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Tear function and ocular surface changes in keratoconus
Tear Function and Ocular Surface Changes in Keratoconus ¨ zc¸etin, MD,1 Haluk Ertu¨rk, MD,1 Murat Dogru, MD, PhD,1,2 Hatice Karakaya, MD,1 Hikmet O 1 1 ¨ zmen, MD, Mehmet Baykara, MD,1 Kazuo Tsubota, MD2 Ali Yu¨cel, MD, Ahmet O Purpose: To describe the ocular surface disorder in patients with keratoconus. Design: A prospective, case-controlled study. Participants: Seventy-five eyes of 38 patients with keratoconus seen at Uludag University School of Medicine, Department of Ophthalmology, from March 2000 through April 2001, and 80 eyes of 40 normal control subjects were studied. Intervention: The subjects underwent routine ophthalmic examinations, corneal sensitivity measurements, Schirmer test, tear film breakup time (BUT), fluorescein and rose bengal staining of the ocular surface, and conjunctival impression cytology. Main Outcome Measures: Patients and control subjects were compared for corneal sensitivity, tear function, ocular surface staining parameters, goblet cell density, and squamous metaplasia grade. The relation of these parameters to the severity of keratoconus progression was also investigated. Results: The mean corneal sensitivity was significantly lower in keratoconus patients compared with control subjects (P ⬍ 0.001). The BUT values were also significantly lower in the keratoconus group. Patients with keratoconus had significantly higher fluorescein and rose bengal staining scores (P ⬍ 0.001). Corneal sensitivity and tear function changes seemed to get worse with advanced stages of keratoconus. Impression cytology showed goblet cell loss and conjunctival squamous metaplasia, both of which again related to the extent of progression of keratoconus. Conclusions: The ocular surface disease in keratoconus is characterized by disorder of tear quality, squamous metaplasia, and goblet cell loss, all of which seem to relate to the extent of keratoconus progression. Ophthalmology 2003;110:1110 –1118 © 2003 by the American Academy of Ophthalmology.
Keratoconus is a progressive, noninflammatory disease of the cornea characterized by cone-shaped protrusion of the corneal surface (ectasia), stromal thinning, irregular corneal astigmatism, and myopia; all of these lead to mild to marked visual impairment. The etiology of the disease remains unknown, although there is evidence of genetic inheritance and possible linkage with systemic disease and circumstantial evidence that certain behaviors, such as excessive eye rubbing and contact lens wear, may be associated with the disease.1–5 Biochemical studies have indicated that the de-
Originally received: November 13, 2001. Accepted: November 5, 2002. Manuscript no. 210971. 1 Uludag University School of Medicine, Department of Ophthalmology, Bursa, Turkey. 2 Tokyo Dental College Ichikawa General Hospital, Department of Ophthalmology, Ichikawa, Japan. Presented at the 30th Congress of the European Contact Lens Society of Ophthalmologists, Antalya, Turkey, September 2000; the 2001 Association for Research in Vision and Ophthalmology meeting, Fort Lauderdale, Florida, April 2001; and the 13th Congress of the European Society of Ophthalmology, Istanbul, Turkey, June 2001. The authors have no proprietary interest in any of the products mentioned in this article. Reprint requests to Murat Dogru, MD, PhD, Department of Ophthalmology, Tokyo Dental College, Ichikawa General Hospital, Sugano 5-11-13, Ichikawa City, Chiba, Japan 272-8513. E-mail: [email protected]
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© 2003 by the American Academy of Ophthalmology Published by Elsevier Inc.
fect of this disease may lie in the degradation processes of the macromolecules in the cornea, with perturbations at the level of corneal epithelium and the stroma.6 –17 Histopathologic studies have pointed out distinct alterations in all corneal layers, depending on the progression of the disease.18 How these alterations relate to tear function and ocular surface changes in keratoconus is still controversial. An increased understanding of the ocular surface disease in keratoconus, including alterations of the tear film and the conjunctival cells, may help explain the pathogenesis of this disorder. Therefore, we performed corneal sensitivity measurements, Schirmer test, tear film breakup time (BUT) analysis, fluorescein and rose bengal staining of the ocular surface, and conjunctival impression cytologic analysis to analyze the relation between the severity of keratoconus and these examinations, and we also compared the results with those of healthy control subjects.
Materials and Methods Subjects and Examinations Seventy-five eyes of 38 keratoconus patients (23 males and 16 females) aged between 12 and 46 years (mean, 24.8 years), as well as 80 eyes of 40 normal subjects aged from 12 to 47 years (mean, 24 years; 20 males and 20 females), were recruited from the Department of Ophthalmology of Uludag University Hospital ISSN 0161-6420/03/$–see front matter doi:10.1016/S0161-6420(03)00261-6
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus Table 1. Progression Assessment of Keratoconus (CLAO) Variable
Corneal Power (D)
Radius of Curvature (mm)
Mild Moderate Severe
⬍45 ⬎45 ⬎52
⬎7.5 ⬍7.5 ⬍6.5
CLAO ⫽ Contact Lens Association of Ophthalmologists; D ⫽ diopters.
from April 2000 through May 2001. Both groups were similar regarding age and sex characteristics. Routine ophthalmic examinations consisted of best-corrected visual acuity measurements, slit-lamp examination, anterior segment photography, corneal topography, keratometry, and pachymetry. At slit-lamp examination, particular attention was paid to lid margins, tarsal and bulbar conjunctiva, and cornea. The instrument used for corneal topography was the Topographic Modeling System (TMS-1; Computed Anatomy, New York, NY). We evaluated topographic data by using the Rabinowitz criteria for the diagnosis of keratoconus; we looked for central corneal power (central K), inferior–superior dioptric asymmetry, and central corneal power differences between the two eyes.2 Corneal topographic patterns were also recorded. The progression assessment of keratoconus was performed by using the guidelines described by the Contact Lens Association of Ophthalmologists and the Japanese Contact Lens Association (Table 1).19 Thus, the stage of keratoconus was graded as mild, moderate, or severe in this study. We obtained pachymetric evidence of corneal thinning by using the Tachy-meter (SonoMed Inc., New York, NY). Pachymetric readings were also obtained from a 6-mm-diameter ring, and the superoinferior corneal thickness difference was calculated from the average of five superior and five inferior points on the ring. Patients with keratoconus who had a history of atopy; allergic diseases; Stevens– Johnson syndrome; chemical, thermal, or radiation injury; or any other ocular or systemic disorder and those who underwent any ocular surgery or contact lens use that would create an ocular surface problem were excluded from this study. The patients and control subjects underwent ocular surface examinations, including corneal sensitivity measurements, tear film BUT, fluorescein and rose bengal staining, Schirmer test, and conjunctival impression cytology. All examinations were performed by the same researcher (HK). Informed consent about the procedures, as well as permission from the Ethical Committee of Uludag University, was obtained. No patient was being treated with topical eye medications at the time of impression cytology. The control subjects did not have any history of ocular or systemic disease or a history of drug or contact lens use that would alter the ocular surface. Measurement of corneal sensitivity was performed with a Cochet–Bonnet aesthesiometer. The measurements were begun with the nylon filament fully extended. The tip of the nylon filament was applied perpendicularly to the surface of the cornea, making certain not to touch the eyelashes, and was pushed until the fiber’s first visible bending. The length of the fiber was gradually decreased until a blink reflex was observed. The length was recorded in millimeters. Measurements were taken from five points, including central, superior, inferior, nasal, and temporal cornea, and the mean of the measurements was recorded as the corneal sensitivity reading of that eye. A corneal sensitivity measurement of ⬍50 mm was regarded as low corneal sensitivity in this study.20,21 The standard tear film BUT measurement was performed. Moistened fluorescein strips were introduced to the conjunctival sac with minimal stimulation and were undetected by the patients.
The subjects were then instructed to blink several times for a few seconds to ensure adequate mixing of fluorescein. The interval between the last complete blink and the appearance of the first corneal black spot in the stained tear film was measured three times, and the mean value of the measurements was calculated. A BUT value of ⬍10 seconds was considered abnormal.22 Fluorescein and rose bengal staining of the cornea was also noted and scored as described elsewhere.23–25 Fluorescein staining scores ranged from 0 to 9 points. A score ⬎3 was regarded as abnormal. Rose bengal staining was performed by using dye-impregnated strips (Akorn Inc., Buffalo Grove, IL) that were wetted by instillation of a single drop of preservative-free tear substitute. The strips were then applied to the conjunctival fornices, and the patients were asked to blink several times. The rose bengal staining scores of the ocular surface ranged from 0 to 9 points. Any score ⬎3 was regarded as abnormal. For further evaluation of tears, the standard Schirmer test with topical anesthesia (0.4% oxybuprocaine chloride) was performed. The standardized strips of filter paper (Alcon Inc., Fort Worth, TX) were placed in the lateral canthus away from the cornea and left in place for 5 minutes with the eyes closed. Readings were reported in millimeters of wetting for 5 minutes. A reading of ⬍5 mm was referred to as dry eye. The impression cytology specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine. Strips of cellulose acetate filter paper (HAWP 304; Millipore, Bedford, MA) that were soaked in distilled water for a few hours and dried at room temperature were applied on the lower nasal bulbar conjunctiva adjacent to the corneal limbus, pressed gently by a glass rod, and then removed. The specimens were then fixed with formaldehyde, stained with periodic acid–Schiff, dehydrated in ascending grades of ethanol and then with xylol, and, finally, coverslipped. The quantitative studies of conjunctival goblet cells and squamous metaplasia of conjunctival epithelial cells were conducted by taking photographs with a calibrated grid under a light microscope at a magnification of 400⫻. We photographed five overlapping areas of each sample selected at random and averaged the outcomes for a single sample score. The goblet cell densities were reported as cells per square millimeter with standard deviations. The specimens were also assigned a grade of conjunctival epithelial squamous metaplasia according to Nelson’s grading scheme.26
Statistical Analysis Data were processed by using Stat View software (Abacus Concepts, Inc., San Diego, CA). The Mann–Whitney U test was used for the analysis of nonparametric values. A probability level ⬍1% was considered statistically significant. The analysis of categorized data was performed with Fisher’s exact probability test, with the probability level set at 1% for statistical significance.
Results Clinical Features There were no age- or sex-related statistical differences between patients with keratoconus and control subjects. The duration of patient-reported keratoconus eye disease varied from 1 to 24 years (mean, 8.0 ⫾ 5.9 years). Thirty-one patients (81.5%) complained of symptoms of eye fatigue, irritation, and foreign-body sensation. Slit-lamp biomicroscopy of the eyelid margins and conjunctiva did not reveal any coexistent blepharitis, meibomian gland disorder, or conjunctivitis. However, 35 eyes (46.6%) had superficial punctate keratopathy. Twenty eyes (57.1%) had diffuse and 15 eyes
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Ophthalmology Volume 110, Number 6, June 2003 Table 2. Slit-Lamp Findings Slit-Lamp Finding Prominent corneal nerves Superficial punctate keratopathy Fleischer’s ring Vogt’s striae Anterior stromal reticular scarring Midstromal scarring Munson’s sign
Table 3. Tear Function and Ocular Surface Examinations Eyes, n (%) 41 (54.6) 35 (46.6) 32 (42.6) 21 (28.0) 16 (21.3) 11 (14.6) 8 (10.6)
Variable Corneal sensitivity (mm) BUT (sec) Schirmer test (mm) Rose bengal score Fluorescein score Squamous metaplasia grade Goblet cell density (cells/mm2)
Patients
Controls
47.0 ⫾ 5.50* 6.80 ⫾ 3.36* 14.6 ⫾ 5.40 3.72 ⫾ 2.35* 3.15 ⫾ 2.19* 1.52 ⫾ 0.75* 470 ⫾ 442*
59.5 ⫾ 0.5 14.5 ⫾ 3.15 16.6 ⫾ 5.1 0.25 ⫾ 0.55 0.05 ⫾ 0.22 0.13 ⫾ 0.35 1181 ⫾ 151
*P ⬍ 0.001, Mann–Whitney U test. BUT ⫽ tear film breakup time.
(42.9%) had inferocentral/interpalpebral superficial punctate keratopathy. None of the control subjects had punctate keratopathy. Prominent corneal nerves were the most commonly observed biomicroscopic signs. Anterior or midstromal scarring was observed in 27 eyes (36%). Munson’s sign was noted in eight eyes (10.6%). Slit-lamp findings are summarized in Table 2.
Keratometric Data There were 3 eyes (4%) with mild keratoconus (central K ⬍ 45 diopters [D]), 24 eyes (31.9%) with moderate keratoconus (45 D ⬍ central K ⬍ 52 D), and 48 eyes (64.1%) with severe keratoconus (central K ⬎ 52 D) in this study.
Topographic Data The mean inferior–superior value was 8.7⫾5.6 D in patients with keratoconus and 1.0 ⫾ 0.5 D in the controls. The mean central corneal power difference between the right and left eyes of keratoconus patients was 5.6 ⫾ 3.5 D, whereas it was 0.5 ⫾ 0.5 D in the control group. Topographical analysis revealed 22 eyes (29.3%) with inferocentral, 40 eyes (53.3%) with inferotemporal, and 13 eyes (17.3%) with central cones.
Pachymetric Data The mean central corneal thicknesses were 452 ⫾ 53 m and 612 ⫾ 23 m in patients with keratoconus and control subjects, respectively. This difference was statistically significant (P ⬍ 0.001). The mean superoinferior corneal thickness difference between the five corresponding points on the 6-mm pachymetry ring was 44.7 ⫾ 18.3 m in the eyes with keratoconus. This value was 10.0 ⫾ 5.0 m in the control subjects. The difference was statistically significant (P ⬍ 0.001).
Corneal Sensitivity Thirty-six eyes (48%) of the patients with keratoconus had low corneal sensitivity. The mean corneal sensitivity in the keratoconus patients was 47.0 ⫾ 5.5 mm, compared with 59.5 ⫾ 0.5 mm in controls (Table 3). This difference was statistically significant (P ⬍ 0.001). The corneal sensitivity was significantly lower in patients with severe keratoconus compared with patients with mild or moderate disease, as shown in Figure 1 (P ⬍ 0.001).
value of ⬍10 seconds, whereas none of the controls had poor performing BUT results. BUT values were significantly lower in patients with moderate and severe keratoconus compared with patients with mild keratoconus, as shown in Figure 2 (P ⬍ 0.001). The Schirmer test value averaged 14.6 ⫾ 5.40 mm in patients with keratoconus vs. 16.6 ⫾ 5.1 mm in control subjects (P ⬎ 0.001). There were no significant differences among patients with mild, moderate, and severe keratoconus in relation to Schirmer test results (Fig 3). Twenty-nine eyes (38.7%) of the patients with keratoconus had a fluorescein staining score ⬎3. The rose bengal staining score was ⬎3 in 54 eyes (72%) in the keratoconus patients. Both fluorescein and rose bengal staining scores were significantly higher in patients with moderate and severe keratoconus compared with controls, as shown in Figure 4 (P ⬍ 0.001). Rose bengal staining over the central cone of a patient with severe keratoconus is shown in Figure 5.
Impression Cytology Conjunctival imprints from healthy control subjects and keratoconus patients contained conjunctival epithelial cells, a variable amount of goblet cells, and mucin. The squamous metaplasia and goblet cell density of each specimen was graded and calculated as described in Materials and Methods. An impression cytology specimen from a healthy control subject is shown in Figure 6.
Squamous Metaplasia The average grade of squamous metaplasia in the keratoconus patients was 1.52 ⫾ 0.75 and was 0.13 ⫾ 0.35 in controls (P ⬍ 0.001). Patients with moderate and severe keratoconus had significantly higher grades of squamous metaplasia compared with patients with mild keratoconus, as shown in Table 4 (P ⬍ 0.001). The impression cytology specimen from the patient with severe keratoconus showed marked loss of cellular cohesion, with isolated epithelial cells in every field and no goblet cells (Fig 7).
Goblet Cell Density The average goblet cell densities were significantly lower in patients with keratoconus when compared with those of the control group (Table 3). Patients with moderate and severe keratoconus had statistically significantly lower goblet cell counts, as seen in Table 4.
Tear Function Parameters The mean BUT in the keratoconus patients was 6.80 ⫾ 3.36 seconds, compared with 14.5 ⫾ 3.15 seconds in controls (Table 3).The difference was statistically significant (P ⬍ 0.001). Fiftythree eyes (70.6%) of the patients with keratoconus had a BUT
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Discussion Previous histopathologic and biochemical studies revealed numerous structural, metabolic, and functional abnormali-
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus
Figure 1. Corneal sensitivity changes with progression of keratoconus.
ties in the conjunctiva and cornea of patients with keratoconus. Recently, release of stromal collagen degradation products (telopeptides) into the tear film of keratoconus patients has also been reported.27 How these abnormalities
affect the ocular surface and the tear functions in keratoconus still remains controversial. We thought that description of the tear film and ocular surface changes in relation to keratoconus progression might provide clues to the pro-
Figure 2. Tear film breakup time (BUT) changes with progression of keratoconus.
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Figure 3. Schirmer test changes with progression of keratoconus.
cesses involved in keratoconus. Thus, we performed corneal sensitivity measurements, Schirmer test, tear film BUT, fluorescein and rose bengal staining of the ocular surface, and conjunctival impression cytology. We analyzed the relation between the extent of keratoconus progression and those examinations and also compared the results with those of normal control subjects. Corneal sensitivity was significantly lower in keratoconus patients compared with controls. We believe that decreased corneal sensitivity in our series of patients strongly implies corneal epithelial/stromal disease. A previous study also showed an overall reduction of corneal sensitivity in all corneal zones of keratoconus patients.28 Indeed, histopathologic evidence from corneal discs obtained at penetrating keratoplasty showed mitochondrial degeneration, liquefaction of neurofibrils, and breaks in the membranes of nerve fibers in the corneal epithelium and the stroma. Most of these changes were especially observed in those nerve fibers that were located in close vicinity to degenerated corneal basal epithelial cells.29 It is interesting to note that we found that corneal sensitivity was significantly reduced in patients with severe keratoconus. The mechanisms of these nerve changes during the course of keratoconus eye disease are still not clear. We believe that loss or decrease of trophic effects of corneal nerves due to primary or secondary events with the progression of keratoconus plays an important role in the pathogenesis of the ocular surface disease in keratoconus. Because we believed that the reduction of corneal sensitivity should have adverse effects on the ocular surface and be able to provide more
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clues, we continued with tear film examinations and ocular surface evaluation by fluorescein and rose bengal staining. Tear function examinations also revealed interesting findings. There were no aqueous-deficient dry eyes in this study. Schirmer test values did not show any significant differences between patients and controls or between patients with mild, moderate, and severe keratoconus. Among our subjects, 70% of the patients had poor BUT scores; 81.5% of the patients reported eye fatigue, irritation, and foreign-body sensation. These findings suggest a BUT-deficient dry-eye state with positive symptomatology as described by Lemp et al.30 Moreover, BUT values were significantly poorer in moderate and severe keratoconus, suggesting an increase of tear film instability with progression of keratoconus. We hypothesized that the reduction of BUT scores resulted from topographic steepening of the cornea, possible alteration of the quality and/or quantity of mucin secretion by the diseased corneal epithelium, reduction of goblet cell numbers, or changes in the conjunctival nongoblet epithelial cells.31–33 The release of collagen degradation products into the tears may also adversely affect the tear film stability. We found that fluorescein and rose bengal scores were significantly higher in keratoconus patients compared with controls. These scores were worse in patients with moderate and severe keratoconus, and this suggests a decline in ocular surface health with the progression of keratoconus. To provide more clues in relation to the tear function changes, we proceeded with impression cytologic analysis, which provided evidence that prominent squamous meta-
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus
Figure 4. Staining score changes with progression of keratoconus.
plasia and goblet cell loss existed in keratoconus patients but not in control subjects. The cytologic changes were more pronounced in patients with severe keratoconus. We also had a noteworthy observation that no mucin was picked up by the filter papers in patients with severe keratoconus. Whether we had a mucin-deficient dry-eye state in keratoconus cannot be answered by this report because we did not quantify the mucin content of tears by using indicators such as hexosamine or O-linked oligosaccharides.34 Such an investigation, along with surface activity determination of aqueous tear components, should provide more answers in relation to mucin deficiency and tear film stability.35,36 In situ hybridization to discern the distribution of changes in mucin gene expression would also provide useful information. Concurrent involvement of the conjunctival and corneal epithelial surfaces in our patients, as evidenced by prominent squamous metaplasia, markedly decreased goblet cell population, and superficial punctate keratopathy may be viewed as an ocular surface disease. Teng29 reported that pathologic changes in the early stages of keratoconus are characterized by fragmentation of the corneal epithelial basement membrane, breaks in Bowman’s membrane, and the death of basal epithelial cells. He noted that liberation of autolytic enzymes from the dead epithelial cells could affect the collagen fibers, keratocytes, nerve fibers, and even Descemet’s membrane. Scanning electron microscopic studies of keratoconus corneas showed that a gradually increasing
number of epithelial cells become detached without being replaced, causing holes and depressions in the corneal epithelial surface, and that in severe cases of keratoconus, part of the epithelium can be denuded of cells, exposing Bowman’s membrane. These studies concluded that there is an accelerated aging process of the corneal epithelial surface.37–39 Others argued that stromal thinning and keratocyte loss in keratoconus can precede the epithelial changes.40 – 42 Moreover, the rarity of recurrent cases of keratoconus in patients who undergo corneal transplantation also argues against the assumption that keratoconus is a disease of epithelial origin.43 It is possible that the development of keratoconus entails several events or requires the participation of multiple factors. Another, more likely, association is that of the changes in the relationship of the cornea and ocular surface to the lids coincident with corneal ectasia. These changes might have altered the surfacing of tears, producing an unstable tear film, poor wetting (particularly over the interpalpebral cornea and conjunctiva), loss of goblet cells, and squamous metaplasia from the drying effect. These events may result in increased corneal staining, which is likely associated with central epithelial breakdown with increased shearing forces from the upper lid, leading to scarring and a decrease in corneal sensation. Whatever the keratoconus-related factors might be, we found marked changes on the ocular surface that affected not only the corneal, but also the conjunctival epithelium. Fukuchi et al44 also noted conjunctival epithelial alterations
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Figure 5. Rose bengal staining over the central cone in a patient with severe keratoconus.
with increased intraepithelial lysosomal enzyme levels. We believe that squamous metaplasia of the conjunctival epithelium may account for some, if not all, of the clinical morbidity of corneal complications in keratoconus. Although further proof is necessary, our impression cytologic findings seemed to strengthen our speculation of ocular surface epithelial involvement in keratoconus. With such an ocular surface epithelial disease hypothesis, the ocular surface epithelium would be diseased in some conditions and not be in jeopardy only because of tear function inadequacies. Treatment in such conditions would thus be directed toward the epithelium. Apart from the conventional dry-eye therapies, including artificial tear drops and lubricants, promising new approaches—such as topical retinoids, which have been shown to promote the production of new basement membrane components and cellular differentiation and to maintain cellular growth, and topical 15-(S)hydroxyeicosatetraenoic acid, which has been shown to cause a rapid-onset increase in mucin secretion by the corneal epithelium—may be promising for the treatment of conjunctival and corneal epithelial disease in keratoconus.45– 47 Impression cytology will help in the decisionmaking to treat such a disease process. However, if the ocular surface disease in keratoconus is genetically programmed to worsen in time with the progression of keratoconus, most treatment measures may remain as palliative measures only. Penetrating keratoplasty with the remote possibility of recurrence would then remain as the best and most successful therapeutic option.2 Recently, altered expression of genes, including ␣1-P-1, acid phosphatase, and
Figure 6. Impression cytology from a healthy subject. Note abundant periodic acid–Schiff–positive oval-plum goblet cells and sheets of small round nonsecretory epithelial cells with a nucleocytoplasmic ratio of 1:1 or 1:2.
cathepsin G, whose products are involved in corneal macromolecular degradation, has been demonstrated. The alteration of these genes as a group suggests that corneal cells, particularly those in the epithelial layer, may exert some form of coordinated transcriptional control to control the characteristic disease phenotype in keratoconus.48,49 We believe that treatment modalities involving gene regulation will be a logical extension of these findings in the future. In conclusion, our data suggest tear function disturbance and ocular surface disease, which seem to evolve in close proximity with the progression of keratoconus. We think that long-term ocular surface studies on the same patients until and even after prescription of contact lenses or a penetrating keratoplasty procedure would provide important information on the natural course of keratoconus ocular surface disease. An investigation into the changes of topographical patterns in time and their correlation with ocular surface disease severity would also be very interesting. We tried to provide some understanding of this disease process and the dry-eye state in this article and emphasized the
Table 4. Impression Cytology Changes with Progression of Keratoconus Variable
Mild
Moderate
Severe
Squamous metaplasia grade Goblet cell density (cells/mm2)
0.77 ⫾ 0.51*
1.50 ⫾ 0.79*
2.0 ⫾ 0.84*
1135 ⫾ 127*
329 ⫾ 225*
135 ⫾ 104*
*P ⬍ 0.001, Fisher’s exact test.
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Figure 7. Impression cytology specimen from the same patient with severe keratoconus, showing marked loss of cellular adhesion, isolated epithelial cells, and no goblet cells.
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus unresolved issues. Thus, it is our strong belief that further work must be initiated and continued along these lines.
References 1. Krachmer JH, Feder RS, Belin MW. Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol 1984;28:293–322. 2. Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998;42:297– 319. 3. Jacobs DS, Dohlman CH. Is keratoconus genetic? Int Ophthalmol Clin 1993;33:249 – 60. 4. Nordan LT. Keratoconus. Diagnosis and treatment. Int Ophthalmol Clin 1997;37:51– 63. 5. Smolek MK, Klyce SD. Is keratoconus a true ectasia? An evaluation of corneal surface area. Arch Ophthalmol 2000; 118:1179 – 86. 6. Critchfield JW, Calandra AJ, Nesburn AB, Kenney MC. Keratoconus. I. Biochemical studies. Exp Eye Res 1988;46:953– 63. 7. Yue BY, Sugar J, Schrode K. Histochemical studies of keratoconus. Curr Eye Res 1988;7:81– 6. 8. Yue BY, Panjwani N, Sugar J, Baum J. Glycoconjugate abnormalities in cultured keratoconus stromal cells. Arch Ophthalmol 1988;106:1709 –12. 9. Brown D, Chwa MM, Opbroek A, Kenney MC. Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors. Curr Eye Res 1993;12:571– 81. 10. Kenney MC, Nesburn AB, Burgeson RE, et al. Abnormalities of the extracellular matrix in keratoconus corneas. Cornea 1997;16:345–51. 11. Tuori A, Virtanen I, Aine E, Uusitalo H. The expression of tenascin and fibronectin in keratoconus, scarred and normal human cornea. Graefes Arch Clin Exp Ophthalmol 1997;235: 222–9. 12. Kao WW, Vergnes JP, Ebert J, et al. Increased collagenase and gelatinase activities in keratoconus. Biochem Biophys Res Commun 1982;107:929 –36. 13. Sawaguchi S, Yue BY, Chang I, et al. Proteoglycan molecules in keratoconus corneas. Invest Ophthalmol Vis Sci 1991;32: 1846 –53. 14. Zhou L, Sawaguchi S, Twining SS, et al. Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci 1998;39:1117–24. 15. Sawaguchi S, Twining SS, Yue BY, et al. Alpha-1 proteinase inhibitor levels in keratoconus. Exp Eye Res 1990;50: 549 –54. 16. Sawaguchi S, Twining SS, Yue BY, et al. ␣2-Macroglobulin levels in normal human and keratoconus corneas. Invest Ophthalmol Vis Sci 1994;35:4008 –14. 17. Daxer A, Fratzl P. Collagen fibril orientation in the human corneal stroma and its implication in keratoconus. Invest Ophthalmol Vis Sci 1997;38:121–9. 18. Kaas-Hansen M. The histopathological changes of keratoconus. Acta Ophthalmol Scand 1993;71:411– 4. 19. Tsubota K. Contact lenses for keratoconus [in Japanese]. In: Tsubota K, ed. Outpatient Clinic of Ophthalmology. Tokyo: Medical View Publishers, 1998;148 –59. 20. Martin XY, Safran AB. Corneal hypoesthesia. Surv Ophthalmol 1988;33:28 – 40. 21. Leibowitz HM, Villazon S. Corneal procedures. In: Leibowitz HM, Waring GO, eds. Corneal Disorders. Philadelphia: WB Saunders, 1998;983– 4.
22. Lemp MA, Dohlman CH, Holly FJ. Corneal desiccation despite normal tear volume. Ann Ophthalmol 1970;6:258 – 84. 23. Pflugfelder SC, Tseng SCG, Yoshino K, et al. Correlation of goblet cell density and mucosal epithelial membrane mucin expression with rose bengal staining in patients with ocular irritation. Ophthalmology 1997;104:223–35. 24. Lee SH, Tseng SC. Rose bengal staining and cytologic characteristics associated with lipid tear deficiency. Am J Ophthalmol 1997;124:736 –50. 25. Junko H. AD classification of fluorescein staining [in Japanese]. In: Tano Y, Watanabe N, eds. Practical Ophthalmology. Tokyo: Bunkodo Publishers, 1998;61. 26. Nelson JD. Impression cytology. Cornea 1988;7:71– 81. 27. Abalain JH, Dossou H, Colin J, Floch HH. Levels of collagen degradation products (telopeptides) in the tear film of patients with keratoconus. Cornea 2000;19:474 – 6. 28. Zabala M, Archila EA. Corneal sensitivity and topogometry in keratoconus. CLAO J 1988;14:210 –2. 29. Teng CC. Electron microscope study of the pathology of keratoconus: part I. Am J Ophthalmol 1963;55:18 – 47. 30. Lemp MA, Dohlman CH, Holly FJ. Corneal desiccation despite normal tear volume. Ann Ophthalmol 1970;6:258 – 84. 31. Kinoshita S, Kiorpes TC, Friend J, Thoft RA. Goblet cell density in ocular surface disease. Arch Ophthalmol 1983;101: 1284 –7. 32. Berry M, Ellingham RB, Corfield AP. Polydispersity of normal human conjunctival mucins. Invest Ophthalmol Vis Sci 1996;37:2559 –71. 33. Jumblatt MM, McKenzie RW, Jumblatt JE. MUC5AC mucin is a component of the human precorneal tear film. Invest Ophthalmol Vis Sci 1999;40:43–9. 34. Dohlman CH, Friend J, Kalevar V, et al. The mucus content of tears from normals and dry eye patients. Exp Eye Res 1976; 22:359 – 65. 35. Holly FJ, Patten JT, Dohlman CH. Surface activity determination of aqueous tear components in dry eye patients and normals. Exp Eye Res 1977;24:479 –91. 36. Holly FJ. Formation and rupture of the tear film. Exp Eye Res 1973;15:515–25. 37. Jongebloed WL, Worst JF. The keratoconus epithelium studied by SEM. Doc Ophthalmol 1987;67:171– 81. 38. Jongebloed WL, Dijk F, Worst JG. Keratoconus morphology and cell dystrophy: a SEM study. Doc Ophthalmol 1989;72: 403–9. 39. Sawaguchi S, Fukuchi T, Abe H, et al. Three dimensional scanning electron microscopic study of keratoconus corneas. Arch Ophthalmol 1998;116:62– 8. 40. Millin JA, Golub BM, Foster CS. Human basement membrane components of keratoconus and normal corneas. Invest Ophthalmol Vis Sci 1986;27:604 –7. 41. Tuori AJ, Virtanen I, Aine E, et al. The immunohistochemical composition of corneal basement membrane in keratoconus. Curr Eye Res 1997;16:792– 801. 42. Scroggs MW, Proia AD. Histopathological variation in keratoconus. Cornea 1992;11:553–9. 43. Bechrakis N, Blom ML, Stark WJ, Green WR. Recurrent keratoconus. Cornea 1994;13:73–7. 44. Fukuchi T, Yue BY, Sugar J, Lam S. Lysosomal enzyme activities in conjunctival tissues of patients with keratoconus. Arch Ophthalmol 1994;112:1368 –74. 45. Tseng SC, Farazdaghi M. Reversal of conjunctival transdifferentiation by topical retinoic acid. Cornea 1988;7:273–9. 46. Tseng SC, Maumenee AE, Stark WJ, et al. Topical retinoid treatment for various dry eye disorders [published erratum
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Ophthalmology Volume 110, Number 6, June 2003 appears in Ophthalmology 1989;96:730]. Ophthalmology 1985;92:717–27. 47. Jackson RS, Van Dyken SJ, McCartney MD, Ubels JL. The eicosanoid, 15-(S)-HETE, stimulates secretion of mucin-like glycoprotein by the corneal epithelium. Cornea 2001;20:516 –21. 48. Whitelock RB, Fukuchi T, Zhou L, et al. Cathepsin G, acid
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phosphatase, and ␣1-proteinase inhibitor messenger RNA levels in keratoconus corneas. Invest Ophthalmol Vis Sci 1997; 38:529 –34. 49. Maruyama Y, Wang X, Li Y, et al. Involvement of Sp1 elements in the promoter activity of genes affected in keratoconus. Invest Ophthalmol Vis Sci 2001;42:1980 –5.
Keratoconus is a progressive, noninflammatory disease of the cornea characterized by cone-shaped protrusion of the corneal surface (ectasia), stromal thinning, irregular corneal astigmatism, and myopia; all of these lead to mild to marked visual impairment. The etiology of the disease remains unknown, although there is evidence of genetic inheritance and possible linkage with systemic disease and circumstantial evidence that certain behaviors, such as excessive eye rubbing and contact lens wear, may be associated with the disease.1–5 Biochemical studies have indicated that the de-
Originally received: November 13, 2001. Accepted: November 5, 2002. Manuscript no. 210971. 1 Uludag University School of Medicine, Department of Ophthalmology, Bursa, Turkey. 2 Tokyo Dental College Ichikawa General Hospital, Department of Ophthalmology, Ichikawa, Japan. Presented at the 30th Congress of the European Contact Lens Society of Ophthalmologists, Antalya, Turkey, September 2000; the 2001 Association for Research in Vision and Ophthalmology meeting, Fort Lauderdale, Florida, April 2001; and the 13th Congress of the European Society of Ophthalmology, Istanbul, Turkey, June 2001. The authors have no proprietary interest in any of the products mentioned in this article. Reprint requests to Murat Dogru, MD, PhD, Department of Ophthalmology, Tokyo Dental College, Ichikawa General Hospital, Sugano 5-11-13, Ichikawa City, Chiba, Japan 272-8513. E-mail: [email protected]
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© 2003 by the American Academy of Ophthalmology Published by Elsevier Inc.
fect of this disease may lie in the degradation processes of the macromolecules in the cornea, with perturbations at the level of corneal epithelium and the stroma.6 –17 Histopathologic studies have pointed out distinct alterations in all corneal layers, depending on the progression of the disease.18 How these alterations relate to tear function and ocular surface changes in keratoconus is still controversial. An increased understanding of the ocular surface disease in keratoconus, including alterations of the tear film and the conjunctival cells, may help explain the pathogenesis of this disorder. Therefore, we performed corneal sensitivity measurements, Schirmer test, tear film breakup time (BUT) analysis, fluorescein and rose bengal staining of the ocular surface, and conjunctival impression cytologic analysis to analyze the relation between the severity of keratoconus and these examinations, and we also compared the results with those of healthy control subjects.
Materials and Methods Subjects and Examinations Seventy-five eyes of 38 keratoconus patients (23 males and 16 females) aged between 12 and 46 years (mean, 24.8 years), as well as 80 eyes of 40 normal subjects aged from 12 to 47 years (mean, 24 years; 20 males and 20 females), were recruited from the Department of Ophthalmology of Uludag University Hospital ISSN 0161-6420/03/$–see front matter doi:10.1016/S0161-6420(03)00261-6
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus Table 1. Progression Assessment of Keratoconus (CLAO) Variable
Corneal Power (D)
Radius of Curvature (mm)
Mild Moderate Severe
⬍45 ⬎45 ⬎52
⬎7.5 ⬍7.5 ⬍6.5
CLAO ⫽ Contact Lens Association of Ophthalmologists; D ⫽ diopters.
from April 2000 through May 2001. Both groups were similar regarding age and sex characteristics. Routine ophthalmic examinations consisted of best-corrected visual acuity measurements, slit-lamp examination, anterior segment photography, corneal topography, keratometry, and pachymetry. At slit-lamp examination, particular attention was paid to lid margins, tarsal and bulbar conjunctiva, and cornea. The instrument used for corneal topography was the Topographic Modeling System (TMS-1; Computed Anatomy, New York, NY). We evaluated topographic data by using the Rabinowitz criteria for the diagnosis of keratoconus; we looked for central corneal power (central K), inferior–superior dioptric asymmetry, and central corneal power differences between the two eyes.2 Corneal topographic patterns were also recorded. The progression assessment of keratoconus was performed by using the guidelines described by the Contact Lens Association of Ophthalmologists and the Japanese Contact Lens Association (Table 1).19 Thus, the stage of keratoconus was graded as mild, moderate, or severe in this study. We obtained pachymetric evidence of corneal thinning by using the Tachy-meter (SonoMed Inc., New York, NY). Pachymetric readings were also obtained from a 6-mm-diameter ring, and the superoinferior corneal thickness difference was calculated from the average of five superior and five inferior points on the ring. Patients with keratoconus who had a history of atopy; allergic diseases; Stevens– Johnson syndrome; chemical, thermal, or radiation injury; or any other ocular or systemic disorder and those who underwent any ocular surgery or contact lens use that would create an ocular surface problem were excluded from this study. The patients and control subjects underwent ocular surface examinations, including corneal sensitivity measurements, tear film BUT, fluorescein and rose bengal staining, Schirmer test, and conjunctival impression cytology. All examinations were performed by the same researcher (HK). Informed consent about the procedures, as well as permission from the Ethical Committee of Uludag University, was obtained. No patient was being treated with topical eye medications at the time of impression cytology. The control subjects did not have any history of ocular or systemic disease or a history of drug or contact lens use that would alter the ocular surface. Measurement of corneal sensitivity was performed with a Cochet–Bonnet aesthesiometer. The measurements were begun with the nylon filament fully extended. The tip of the nylon filament was applied perpendicularly to the surface of the cornea, making certain not to touch the eyelashes, and was pushed until the fiber’s first visible bending. The length of the fiber was gradually decreased until a blink reflex was observed. The length was recorded in millimeters. Measurements were taken from five points, including central, superior, inferior, nasal, and temporal cornea, and the mean of the measurements was recorded as the corneal sensitivity reading of that eye. A corneal sensitivity measurement of ⬍50 mm was regarded as low corneal sensitivity in this study.20,21 The standard tear film BUT measurement was performed. Moistened fluorescein strips were introduced to the conjunctival sac with minimal stimulation and were undetected by the patients.
The subjects were then instructed to blink several times for a few seconds to ensure adequate mixing of fluorescein. The interval between the last complete blink and the appearance of the first corneal black spot in the stained tear film was measured three times, and the mean value of the measurements was calculated. A BUT value of ⬍10 seconds was considered abnormal.22 Fluorescein and rose bengal staining of the cornea was also noted and scored as described elsewhere.23–25 Fluorescein staining scores ranged from 0 to 9 points. A score ⬎3 was regarded as abnormal. Rose bengal staining was performed by using dye-impregnated strips (Akorn Inc., Buffalo Grove, IL) that were wetted by instillation of a single drop of preservative-free tear substitute. The strips were then applied to the conjunctival fornices, and the patients were asked to blink several times. The rose bengal staining scores of the ocular surface ranged from 0 to 9 points. Any score ⬎3 was regarded as abnormal. For further evaluation of tears, the standard Schirmer test with topical anesthesia (0.4% oxybuprocaine chloride) was performed. The standardized strips of filter paper (Alcon Inc., Fort Worth, TX) were placed in the lateral canthus away from the cornea and left in place for 5 minutes with the eyes closed. Readings were reported in millimeters of wetting for 5 minutes. A reading of ⬍5 mm was referred to as dry eye. The impression cytology specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine. Strips of cellulose acetate filter paper (HAWP 304; Millipore, Bedford, MA) that were soaked in distilled water for a few hours and dried at room temperature were applied on the lower nasal bulbar conjunctiva adjacent to the corneal limbus, pressed gently by a glass rod, and then removed. The specimens were then fixed with formaldehyde, stained with periodic acid–Schiff, dehydrated in ascending grades of ethanol and then with xylol, and, finally, coverslipped. The quantitative studies of conjunctival goblet cells and squamous metaplasia of conjunctival epithelial cells were conducted by taking photographs with a calibrated grid under a light microscope at a magnification of 400⫻. We photographed five overlapping areas of each sample selected at random and averaged the outcomes for a single sample score. The goblet cell densities were reported as cells per square millimeter with standard deviations. The specimens were also assigned a grade of conjunctival epithelial squamous metaplasia according to Nelson’s grading scheme.26
Statistical Analysis Data were processed by using Stat View software (Abacus Concepts, Inc., San Diego, CA). The Mann–Whitney U test was used for the analysis of nonparametric values. A probability level ⬍1% was considered statistically significant. The analysis of categorized data was performed with Fisher’s exact probability test, with the probability level set at 1% for statistical significance.
Results Clinical Features There were no age- or sex-related statistical differences between patients with keratoconus and control subjects. The duration of patient-reported keratoconus eye disease varied from 1 to 24 years (mean, 8.0 ⫾ 5.9 years). Thirty-one patients (81.5%) complained of symptoms of eye fatigue, irritation, and foreign-body sensation. Slit-lamp biomicroscopy of the eyelid margins and conjunctiva did not reveal any coexistent blepharitis, meibomian gland disorder, or conjunctivitis. However, 35 eyes (46.6%) had superficial punctate keratopathy. Twenty eyes (57.1%) had diffuse and 15 eyes
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Ophthalmology Volume 110, Number 6, June 2003 Table 2. Slit-Lamp Findings Slit-Lamp Finding Prominent corneal nerves Superficial punctate keratopathy Fleischer’s ring Vogt’s striae Anterior stromal reticular scarring Midstromal scarring Munson’s sign
Table 3. Tear Function and Ocular Surface Examinations Eyes, n (%) 41 (54.6) 35 (46.6) 32 (42.6) 21 (28.0) 16 (21.3) 11 (14.6) 8 (10.6)
Variable Corneal sensitivity (mm) BUT (sec) Schirmer test (mm) Rose bengal score Fluorescein score Squamous metaplasia grade Goblet cell density (cells/mm2)
Patients
Controls
47.0 ⫾ 5.50* 6.80 ⫾ 3.36* 14.6 ⫾ 5.40 3.72 ⫾ 2.35* 3.15 ⫾ 2.19* 1.52 ⫾ 0.75* 470 ⫾ 442*
59.5 ⫾ 0.5 14.5 ⫾ 3.15 16.6 ⫾ 5.1 0.25 ⫾ 0.55 0.05 ⫾ 0.22 0.13 ⫾ 0.35 1181 ⫾ 151
*P ⬍ 0.001, Mann–Whitney U test. BUT ⫽ tear film breakup time.
(42.9%) had inferocentral/interpalpebral superficial punctate keratopathy. None of the control subjects had punctate keratopathy. Prominent corneal nerves were the most commonly observed biomicroscopic signs. Anterior or midstromal scarring was observed in 27 eyes (36%). Munson’s sign was noted in eight eyes (10.6%). Slit-lamp findings are summarized in Table 2.
Keratometric Data There were 3 eyes (4%) with mild keratoconus (central K ⬍ 45 diopters [D]), 24 eyes (31.9%) with moderate keratoconus (45 D ⬍ central K ⬍ 52 D), and 48 eyes (64.1%) with severe keratoconus (central K ⬎ 52 D) in this study.
Topographic Data The mean inferior–superior value was 8.7⫾5.6 D in patients with keratoconus and 1.0 ⫾ 0.5 D in the controls. The mean central corneal power difference between the right and left eyes of keratoconus patients was 5.6 ⫾ 3.5 D, whereas it was 0.5 ⫾ 0.5 D in the control group. Topographical analysis revealed 22 eyes (29.3%) with inferocentral, 40 eyes (53.3%) with inferotemporal, and 13 eyes (17.3%) with central cones.
Pachymetric Data The mean central corneal thicknesses were 452 ⫾ 53 m and 612 ⫾ 23 m in patients with keratoconus and control subjects, respectively. This difference was statistically significant (P ⬍ 0.001). The mean superoinferior corneal thickness difference between the five corresponding points on the 6-mm pachymetry ring was 44.7 ⫾ 18.3 m in the eyes with keratoconus. This value was 10.0 ⫾ 5.0 m in the control subjects. The difference was statistically significant (P ⬍ 0.001).
Corneal Sensitivity Thirty-six eyes (48%) of the patients with keratoconus had low corneal sensitivity. The mean corneal sensitivity in the keratoconus patients was 47.0 ⫾ 5.5 mm, compared with 59.5 ⫾ 0.5 mm in controls (Table 3). This difference was statistically significant (P ⬍ 0.001). The corneal sensitivity was significantly lower in patients with severe keratoconus compared with patients with mild or moderate disease, as shown in Figure 1 (P ⬍ 0.001).
value of ⬍10 seconds, whereas none of the controls had poor performing BUT results. BUT values were significantly lower in patients with moderate and severe keratoconus compared with patients with mild keratoconus, as shown in Figure 2 (P ⬍ 0.001). The Schirmer test value averaged 14.6 ⫾ 5.40 mm in patients with keratoconus vs. 16.6 ⫾ 5.1 mm in control subjects (P ⬎ 0.001). There were no significant differences among patients with mild, moderate, and severe keratoconus in relation to Schirmer test results (Fig 3). Twenty-nine eyes (38.7%) of the patients with keratoconus had a fluorescein staining score ⬎3. The rose bengal staining score was ⬎3 in 54 eyes (72%) in the keratoconus patients. Both fluorescein and rose bengal staining scores were significantly higher in patients with moderate and severe keratoconus compared with controls, as shown in Figure 4 (P ⬍ 0.001). Rose bengal staining over the central cone of a patient with severe keratoconus is shown in Figure 5.
Impression Cytology Conjunctival imprints from healthy control subjects and keratoconus patients contained conjunctival epithelial cells, a variable amount of goblet cells, and mucin. The squamous metaplasia and goblet cell density of each specimen was graded and calculated as described in Materials and Methods. An impression cytology specimen from a healthy control subject is shown in Figure 6.
Squamous Metaplasia The average grade of squamous metaplasia in the keratoconus patients was 1.52 ⫾ 0.75 and was 0.13 ⫾ 0.35 in controls (P ⬍ 0.001). Patients with moderate and severe keratoconus had significantly higher grades of squamous metaplasia compared with patients with mild keratoconus, as shown in Table 4 (P ⬍ 0.001). The impression cytology specimen from the patient with severe keratoconus showed marked loss of cellular cohesion, with isolated epithelial cells in every field and no goblet cells (Fig 7).
Goblet Cell Density The average goblet cell densities were significantly lower in patients with keratoconus when compared with those of the control group (Table 3). Patients with moderate and severe keratoconus had statistically significantly lower goblet cell counts, as seen in Table 4.
Tear Function Parameters The mean BUT in the keratoconus patients was 6.80 ⫾ 3.36 seconds, compared with 14.5 ⫾ 3.15 seconds in controls (Table 3).The difference was statistically significant (P ⬍ 0.001). Fiftythree eyes (70.6%) of the patients with keratoconus had a BUT
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Discussion Previous histopathologic and biochemical studies revealed numerous structural, metabolic, and functional abnormali-
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus
Figure 1. Corneal sensitivity changes with progression of keratoconus.
ties in the conjunctiva and cornea of patients with keratoconus. Recently, release of stromal collagen degradation products (telopeptides) into the tear film of keratoconus patients has also been reported.27 How these abnormalities
affect the ocular surface and the tear functions in keratoconus still remains controversial. We thought that description of the tear film and ocular surface changes in relation to keratoconus progression might provide clues to the pro-
Figure 2. Tear film breakup time (BUT) changes with progression of keratoconus.
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Figure 3. Schirmer test changes with progression of keratoconus.
cesses involved in keratoconus. Thus, we performed corneal sensitivity measurements, Schirmer test, tear film BUT, fluorescein and rose bengal staining of the ocular surface, and conjunctival impression cytology. We analyzed the relation between the extent of keratoconus progression and those examinations and also compared the results with those of normal control subjects. Corneal sensitivity was significantly lower in keratoconus patients compared with controls. We believe that decreased corneal sensitivity in our series of patients strongly implies corneal epithelial/stromal disease. A previous study also showed an overall reduction of corneal sensitivity in all corneal zones of keratoconus patients.28 Indeed, histopathologic evidence from corneal discs obtained at penetrating keratoplasty showed mitochondrial degeneration, liquefaction of neurofibrils, and breaks in the membranes of nerve fibers in the corneal epithelium and the stroma. Most of these changes were especially observed in those nerve fibers that were located in close vicinity to degenerated corneal basal epithelial cells.29 It is interesting to note that we found that corneal sensitivity was significantly reduced in patients with severe keratoconus. The mechanisms of these nerve changes during the course of keratoconus eye disease are still not clear. We believe that loss or decrease of trophic effects of corneal nerves due to primary or secondary events with the progression of keratoconus plays an important role in the pathogenesis of the ocular surface disease in keratoconus. Because we believed that the reduction of corneal sensitivity should have adverse effects on the ocular surface and be able to provide more
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clues, we continued with tear film examinations and ocular surface evaluation by fluorescein and rose bengal staining. Tear function examinations also revealed interesting findings. There were no aqueous-deficient dry eyes in this study. Schirmer test values did not show any significant differences between patients and controls or between patients with mild, moderate, and severe keratoconus. Among our subjects, 70% of the patients had poor BUT scores; 81.5% of the patients reported eye fatigue, irritation, and foreign-body sensation. These findings suggest a BUT-deficient dry-eye state with positive symptomatology as described by Lemp et al.30 Moreover, BUT values were significantly poorer in moderate and severe keratoconus, suggesting an increase of tear film instability with progression of keratoconus. We hypothesized that the reduction of BUT scores resulted from topographic steepening of the cornea, possible alteration of the quality and/or quantity of mucin secretion by the diseased corneal epithelium, reduction of goblet cell numbers, or changes in the conjunctival nongoblet epithelial cells.31–33 The release of collagen degradation products into the tears may also adversely affect the tear film stability. We found that fluorescein and rose bengal scores were significantly higher in keratoconus patients compared with controls. These scores were worse in patients with moderate and severe keratoconus, and this suggests a decline in ocular surface health with the progression of keratoconus. To provide more clues in relation to the tear function changes, we proceeded with impression cytologic analysis, which provided evidence that prominent squamous meta-
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus
Figure 4. Staining score changes with progression of keratoconus.
plasia and goblet cell loss existed in keratoconus patients but not in control subjects. The cytologic changes were more pronounced in patients with severe keratoconus. We also had a noteworthy observation that no mucin was picked up by the filter papers in patients with severe keratoconus. Whether we had a mucin-deficient dry-eye state in keratoconus cannot be answered by this report because we did not quantify the mucin content of tears by using indicators such as hexosamine or O-linked oligosaccharides.34 Such an investigation, along with surface activity determination of aqueous tear components, should provide more answers in relation to mucin deficiency and tear film stability.35,36 In situ hybridization to discern the distribution of changes in mucin gene expression would also provide useful information. Concurrent involvement of the conjunctival and corneal epithelial surfaces in our patients, as evidenced by prominent squamous metaplasia, markedly decreased goblet cell population, and superficial punctate keratopathy may be viewed as an ocular surface disease. Teng29 reported that pathologic changes in the early stages of keratoconus are characterized by fragmentation of the corneal epithelial basement membrane, breaks in Bowman’s membrane, and the death of basal epithelial cells. He noted that liberation of autolytic enzymes from the dead epithelial cells could affect the collagen fibers, keratocytes, nerve fibers, and even Descemet’s membrane. Scanning electron microscopic studies of keratoconus corneas showed that a gradually increasing
number of epithelial cells become detached without being replaced, causing holes and depressions in the corneal epithelial surface, and that in severe cases of keratoconus, part of the epithelium can be denuded of cells, exposing Bowman’s membrane. These studies concluded that there is an accelerated aging process of the corneal epithelial surface.37–39 Others argued that stromal thinning and keratocyte loss in keratoconus can precede the epithelial changes.40 – 42 Moreover, the rarity of recurrent cases of keratoconus in patients who undergo corneal transplantation also argues against the assumption that keratoconus is a disease of epithelial origin.43 It is possible that the development of keratoconus entails several events or requires the participation of multiple factors. Another, more likely, association is that of the changes in the relationship of the cornea and ocular surface to the lids coincident with corneal ectasia. These changes might have altered the surfacing of tears, producing an unstable tear film, poor wetting (particularly over the interpalpebral cornea and conjunctiva), loss of goblet cells, and squamous metaplasia from the drying effect. These events may result in increased corneal staining, which is likely associated with central epithelial breakdown with increased shearing forces from the upper lid, leading to scarring and a decrease in corneal sensation. Whatever the keratoconus-related factors might be, we found marked changes on the ocular surface that affected not only the corneal, but also the conjunctival epithelium. Fukuchi et al44 also noted conjunctival epithelial alterations
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Figure 5. Rose bengal staining over the central cone in a patient with severe keratoconus.
with increased intraepithelial lysosomal enzyme levels. We believe that squamous metaplasia of the conjunctival epithelium may account for some, if not all, of the clinical morbidity of corneal complications in keratoconus. Although further proof is necessary, our impression cytologic findings seemed to strengthen our speculation of ocular surface epithelial involvement in keratoconus. With such an ocular surface epithelial disease hypothesis, the ocular surface epithelium would be diseased in some conditions and not be in jeopardy only because of tear function inadequacies. Treatment in such conditions would thus be directed toward the epithelium. Apart from the conventional dry-eye therapies, including artificial tear drops and lubricants, promising new approaches—such as topical retinoids, which have been shown to promote the production of new basement membrane components and cellular differentiation and to maintain cellular growth, and topical 15-(S)hydroxyeicosatetraenoic acid, which has been shown to cause a rapid-onset increase in mucin secretion by the corneal epithelium—may be promising for the treatment of conjunctival and corneal epithelial disease in keratoconus.45– 47 Impression cytology will help in the decisionmaking to treat such a disease process. However, if the ocular surface disease in keratoconus is genetically programmed to worsen in time with the progression of keratoconus, most treatment measures may remain as palliative measures only. Penetrating keratoplasty with the remote possibility of recurrence would then remain as the best and most successful therapeutic option.2 Recently, altered expression of genes, including ␣1-P-1, acid phosphatase, and
Figure 6. Impression cytology from a healthy subject. Note abundant periodic acid–Schiff–positive oval-plum goblet cells and sheets of small round nonsecretory epithelial cells with a nucleocytoplasmic ratio of 1:1 or 1:2.
cathepsin G, whose products are involved in corneal macromolecular degradation, has been demonstrated. The alteration of these genes as a group suggests that corneal cells, particularly those in the epithelial layer, may exert some form of coordinated transcriptional control to control the characteristic disease phenotype in keratoconus.48,49 We believe that treatment modalities involving gene regulation will be a logical extension of these findings in the future. In conclusion, our data suggest tear function disturbance and ocular surface disease, which seem to evolve in close proximity with the progression of keratoconus. We think that long-term ocular surface studies on the same patients until and even after prescription of contact lenses or a penetrating keratoplasty procedure would provide important information on the natural course of keratoconus ocular surface disease. An investigation into the changes of topographical patterns in time and their correlation with ocular surface disease severity would also be very interesting. We tried to provide some understanding of this disease process and the dry-eye state in this article and emphasized the
Table 4. Impression Cytology Changes with Progression of Keratoconus Variable
Mild
Moderate
Severe
Squamous metaplasia grade Goblet cell density (cells/mm2)
0.77 ⫾ 0.51*
1.50 ⫾ 0.79*
2.0 ⫾ 0.84*
1135 ⫾ 127*
329 ⫾ 225*
135 ⫾ 104*
*P ⬍ 0.001, Fisher’s exact test.
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Figure 7. Impression cytology specimen from the same patient with severe keratoconus, showing marked loss of cellular adhesion, isolated epithelial cells, and no goblet cells.
Dogru et al 䡠 Tear Function and Ocular Surface Changes in Keratoconus unresolved issues. Thus, it is our strong belief that further work must be initiated and continued along these lines.
References 1. Krachmer JH, Feder RS, Belin MW. Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol 1984;28:293–322. 2. Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998;42:297– 319. 3. Jacobs DS, Dohlman CH. Is keratoconus genetic? Int Ophthalmol Clin 1993;33:249 – 60. 4. Nordan LT. Keratoconus. Diagnosis and treatment. Int Ophthalmol Clin 1997;37:51– 63. 5. Smolek MK, Klyce SD. Is keratoconus a true ectasia? An evaluation of corneal surface area. Arch Ophthalmol 2000; 118:1179 – 86. 6. Critchfield JW, Calandra AJ, Nesburn AB, Kenney MC. Keratoconus. I. Biochemical studies. Exp Eye Res 1988;46:953– 63. 7. Yue BY, Sugar J, Schrode K. Histochemical studies of keratoconus. Curr Eye Res 1988;7:81– 6. 8. Yue BY, Panjwani N, Sugar J, Baum J. Glycoconjugate abnormalities in cultured keratoconus stromal cells. Arch Ophthalmol 1988;106:1709 –12. 9. Brown D, Chwa MM, Opbroek A, Kenney MC. Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors. Curr Eye Res 1993;12:571– 81. 10. Kenney MC, Nesburn AB, Burgeson RE, et al. Abnormalities of the extracellular matrix in keratoconus corneas. Cornea 1997;16:345–51. 11. Tuori A, Virtanen I, Aine E, Uusitalo H. The expression of tenascin and fibronectin in keratoconus, scarred and normal human cornea. Graefes Arch Clin Exp Ophthalmol 1997;235: 222–9. 12. Kao WW, Vergnes JP, Ebert J, et al. Increased collagenase and gelatinase activities in keratoconus. Biochem Biophys Res Commun 1982;107:929 –36. 13. Sawaguchi S, Yue BY, Chang I, et al. Proteoglycan molecules in keratoconus corneas. Invest Ophthalmol Vis Sci 1991;32: 1846 –53. 14. Zhou L, Sawaguchi S, Twining SS, et al. Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci 1998;39:1117–24. 15. Sawaguchi S, Twining SS, Yue BY, et al. Alpha-1 proteinase inhibitor levels in keratoconus. Exp Eye Res 1990;50: 549 –54. 16. Sawaguchi S, Twining SS, Yue BY, et al. ␣2-Macroglobulin levels in normal human and keratoconus corneas. Invest Ophthalmol Vis Sci 1994;35:4008 –14. 17. Daxer A, Fratzl P. Collagen fibril orientation in the human corneal stroma and its implication in keratoconus. Invest Ophthalmol Vis Sci 1997;38:121–9. 18. Kaas-Hansen M. The histopathological changes of keratoconus. Acta Ophthalmol Scand 1993;71:411– 4. 19. Tsubota K. Contact lenses for keratoconus [in Japanese]. In: Tsubota K, ed. Outpatient Clinic of Ophthalmology. Tokyo: Medical View Publishers, 1998;148 –59. 20. Martin XY, Safran AB. Corneal hypoesthesia. Surv Ophthalmol 1988;33:28 – 40. 21. Leibowitz HM, Villazon S. Corneal procedures. In: Leibowitz HM, Waring GO, eds. Corneal Disorders. Philadelphia: WB Saunders, 1998;983– 4.
22. Lemp MA, Dohlman CH, Holly FJ. Corneal desiccation despite normal tear volume. Ann Ophthalmol 1970;6:258 – 84. 23. Pflugfelder SC, Tseng SCG, Yoshino K, et al. Correlation of goblet cell density and mucosal epithelial membrane mucin expression with rose bengal staining in patients with ocular irritation. Ophthalmology 1997;104:223–35. 24. Lee SH, Tseng SC. Rose bengal staining and cytologic characteristics associated with lipid tear deficiency. Am J Ophthalmol 1997;124:736 –50. 25. Junko H. AD classification of fluorescein staining [in Japanese]. In: Tano Y, Watanabe N, eds. Practical Ophthalmology. Tokyo: Bunkodo Publishers, 1998;61. 26. Nelson JD. Impression cytology. Cornea 1988;7:71– 81. 27. Abalain JH, Dossou H, Colin J, Floch HH. Levels of collagen degradation products (telopeptides) in the tear film of patients with keratoconus. Cornea 2000;19:474 – 6. 28. Zabala M, Archila EA. Corneal sensitivity and topogometry in keratoconus. CLAO J 1988;14:210 –2. 29. Teng CC. Electron microscope study of the pathology of keratoconus: part I. Am J Ophthalmol 1963;55:18 – 47. 30. Lemp MA, Dohlman CH, Holly FJ. Corneal desiccation despite normal tear volume. Ann Ophthalmol 1970;6:258 – 84. 31. Kinoshita S, Kiorpes TC, Friend J, Thoft RA. Goblet cell density in ocular surface disease. Arch Ophthalmol 1983;101: 1284 –7. 32. Berry M, Ellingham RB, Corfield AP. Polydispersity of normal human conjunctival mucins. Invest Ophthalmol Vis Sci 1996;37:2559 –71. 33. Jumblatt MM, McKenzie RW, Jumblatt JE. MUC5AC mucin is a component of the human precorneal tear film. Invest Ophthalmol Vis Sci 1999;40:43–9. 34. Dohlman CH, Friend J, Kalevar V, et al. The mucus content of tears from normals and dry eye patients. Exp Eye Res 1976; 22:359 – 65. 35. Holly FJ, Patten JT, Dohlman CH. Surface activity determination of aqueous tear components in dry eye patients and normals. Exp Eye Res 1977;24:479 –91. 36. Holly FJ. Formation and rupture of the tear film. Exp Eye Res 1973;15:515–25. 37. Jongebloed WL, Worst JF. The keratoconus epithelium studied by SEM. Doc Ophthalmol 1987;67:171– 81. 38. Jongebloed WL, Dijk F, Worst JG. Keratoconus morphology and cell dystrophy: a SEM study. Doc Ophthalmol 1989;72: 403–9. 39. Sawaguchi S, Fukuchi T, Abe H, et al. Three dimensional scanning electron microscopic study of keratoconus corneas. Arch Ophthalmol 1998;116:62– 8. 40. Millin JA, Golub BM, Foster CS. Human basement membrane components of keratoconus and normal corneas. Invest Ophthalmol Vis Sci 1986;27:604 –7. 41. Tuori AJ, Virtanen I, Aine E, et al. The immunohistochemical composition of corneal basement membrane in keratoconus. Curr Eye Res 1997;16:792– 801. 42. Scroggs MW, Proia AD. Histopathological variation in keratoconus. Cornea 1992;11:553–9. 43. Bechrakis N, Blom ML, Stark WJ, Green WR. Recurrent keratoconus. Cornea 1994;13:73–7. 44. Fukuchi T, Yue BY, Sugar J, Lam S. Lysosomal enzyme activities in conjunctival tissues of patients with keratoconus. Arch Ophthalmol 1994;112:1368 –74. 45. Tseng SC, Farazdaghi M. Reversal of conjunctival transdifferentiation by topical retinoic acid. Cornea 1988;7:273–9. 46. Tseng SC, Maumenee AE, Stark WJ, et al. Topical retinoid treatment for various dry eye disorders [published erratum
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Ophthalmology Volume 110, Number 6, June 2003 appears in Ophthalmology 1989;96:730]. Ophthalmology 1985;92:717–27. 47. Jackson RS, Van Dyken SJ, McCartney MD, Ubels JL. The eicosanoid, 15-(S)-HETE, stimulates secretion of mucin-like glycoprotein by the corneal epithelium. Cornea 2001;20:516 –21. 48. Whitelock RB, Fukuchi T, Zhou L, et al. Cathepsin G, acid
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phosphatase, and ␣1-proteinase inhibitor messenger RNA levels in keratoconus corneas. Invest Ophthalmol Vis Sci 1997; 38:529 –34. 49. Maruyama Y, Wang X, Li Y, et al. Involvement of Sp1 elements in the promoter activity of genes affected in keratoconus. Invest Ophthalmol Vis Sci 2001;42:1980 –5.