Current Eye Research, Early Online, 1–7, 2014 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.987871
RESEARCH REPORT
Signs and Symptoms of Dry Eye in Keratoconus Patients: A Pilot Study Gonzalo Carracedo1, Alberto Recchioni1, Nicola´s Alejandre-Alba2, Alba Martin-Gil3, Almudena Crooke3, Ignacio Jimenez-Alfaro Morote2 and Jesu´s Pintor3
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1
Department of Optics II, Optometry and Vision, Faculty of Optics, Universidad Complutense de Madrid, Madrid, Spain, 2Department of Ophthalmology, Fundacio´n Jimenez Diaz, Madrid, Spain, and 3Department of Biochemistry and Molecular Biology IV, Faculty of Optics, Universidad Complutense de Madrid, Madrid, Spain
ABSTRACT Purpose: To compare signs and symptoms of dry eye in keratoconus (KC) patients versus healthy subjects. Methods: A total of 15 KC patients (KC group, n = 15 eyes) and 16 healthy subjects (control group, 16 eyes) were enrolled in this study. The Schirmer I test with no anesthetic, tear break-up time (TBUT), corneal staining characteristics, and ocular surface disease index (OSDI) scores were evaluated for both groups. Impression cytology, combined with/scanning laser confocal microscopy (LCM), was performed to evaluate goblet cell density, mucin cloud height (MCH), and goblet cell layer thickness (CLT). Finally, tear concentrations of di-adenosine tetraphosphate (Ap4A) were assessed. Results were statistically analyzed using Shapiro–Wilk and non-parametric Wilcoxon rank sum tests. Statistical significance was set at p50.05. Results: KC patients had lower tear volumes and greater corneal staining than did healthy subjects (p50.05). OSDI scores were 44.96 ± 8.65 and 17.78 ± 6.50 for the KC and control groups, respectively (p50.05). We found no statistically significant differences in TBUT between groups. Impression cytology revealed lower goblet cell densities in KC group patients versus control group subjects (84.88 ± 32.98 and 128.88 ± 50.60 cells/mm,2 respectively, p50.05). There was a statistically significant reduction in MCH and CLT in KC group patients compared with control group subjects. Ap4A tear concentrations were higher in KC group patients than in control group subjects (2.56 ± 1.10 and 0.15 ± 0.12 mM, respectively, p50.05). Conclusions: The parameters evaluated in this study indicate that KC patients suffer greater symptoms of dry eye and greater tear instability, primarily due to the decreased mucin production in their tears, than do healthy patients with no KC Keywords: 3D impression cytology, dinucleotide, dry eye, keratoconus
INTRODUCTION
presents between the first and the third decades of life, and is rarely congenital.6 The prevalence of the disease is 0.2–2.3% of the population.7–10 The multifactorial etiology of KC includes genetic factors,11 allergy,12 UV light exposure, and eye rubbing. Eye rubbing, which has been reported in 40–70% of keratoconus patients,13 is an event that is stimulated by ocular discomfort, i.e. burning, itching, and dry eyes.14 A recent clinical study demonstrated reduced tear quality, as evidenced by a reduced tear
Keratoconus (KC) is an ectatic disease of the central and paracentral cornea. The disease primarily not only involves Bowman’s membrane but also includes the anterior and middle stroma.1,2 Keratoconus creates alterations of the corneal surface that result in visual distortion, reduced tear film quality,3 and ocular discomfort.4 Keratoconus is a bilateral, progressive, and asymmetric disorder.5 It initially
Received 13 December 2013; revised 4 November 2014; accepted 9 November 2014; published online 9 December 2014 Correspondence: Gonzalo Carracedo, Department Optics II (Optometry and Vision), Faculty of Optics, Universidad Complutense de Madrid, C/Arcos del Jalon 118, 28032 Madrid, Spain. E-mail: [email protected]
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G. Carracedo et al. TABLE 1 Demographics. Parameter
Keratoconus group
Control group
p Value
No. eyes (no. patients) Mean age (years) ± SD Age range (years) Gender (male, female) Mean keratometry (D) This not mentioned in manuscript Flat Steep
15 (15) 39.30 ± 6.49 18–44 8, 7
16 (16) 26.56 ± 6.50 20–34 8, 8
– 50.001 – –
49.46 ± 6.32 56.35 ± 8.76
43.87 ± 2.53 45.31 ± 1.39
0.003 50.001
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p50.05, Wilcoxon Rank Sum test.
break-up time (TBUT), and increased corneal staining in KC patients.3 Biomicroscoy of KC patients who wear rigid gas permeable (RGP) contact lenses often reveals signs of mechanical trauma to the lens. This, in combination with frequent eye rubbing, typically leads to a strong inflammatory response – interleukin (IL) 1-a and -b – in the corneal epithelium. This inflammation, in turn, induces apoptosis of corneal stromal cells and fibroblasts.15–17 Because the ocular surface signs of KC are similar to those of dry eye syndrome (DES), we investigated the potential relationship between KC and dry eye. Impression cytology, considered the gold standard for diagnosing DES,18 assesses the density of the mucin-producing conjunctival goblet cells, which are critical for maintaining ocular surface integrity.19 A recently developed LCM technique for analyzing impression cytology allows 3D imaging of goblet cell density, CLT, and mucin cloud (MC) height.20 Using this combination technique, Peral and Pintor20 discerned that a number of goblet cells were not secreting mucins. Impression cytology/LCM thus provides more meaningful and objective details for diagnosis that previously available. Besides goblet cell analysis, testing for other substances present in tears can serve as markers of ocular surface dryness. One such component is di-adenosine tetraphosphate (Ap4A), which has been proposed as being a potential molecular biomarker for DES.21 In symptomatic, normal tear-producing, non-Sjo¨gren dry-eye patients, Ap4A levels were shown to be increased five-fold and were almost 100 times greater than in symptomatic non-Sjo¨gren dry-eye patients with low tear production. Additionally, Ap4A concentrations were increased to 42-fold in Sjo¨gren dry-eye patients compared with normal individuals.22 To the best of our knowledge, no previous studies have correlated symptoms of DES with objective measures, e.g., Ap4A concentrations or goblet cell3D analysis, in KC patients. Thus, the objective of this study was to evaluate if keratoconus patients have more signs and symptoms of DES than do nonkeratoconus patients.
MATERIALS AND METHODS This pilot, experimental, prospective study included 15 keratoconus subjects (eight men and seven women), mean ages of 39.30 ± 6.49 (range, 18–44) years old and 16 healthy subjects (eight men and eight women), mean ages of 26.56 ± 6.50 (range, 20–34) years, all of had been treated at the Fundacio´n Jime´nez-Dı´az, Madrid, Spain, and the University Clinic of Optometry, Complutense University, Madrid, Spain. Subjects already diagnosed with DES or subjects undergoing treatment for DES were excluded. All subjects took part voluntarily in the study and were free to leave without question if they chose. All subjects provided informed consent after, the nature of the research and the risks of participation were explained. The study adhered to the tenets of the Declaration of Helsinki23 and was approved by the Ethics Committee (CEIC) of the Fundacio´n Jime´nez-Dı´az. Detailed demographic characteristics of the study population are shown in Table 1. All subjects were contact lens wearers, except two control subjects who had never worn contact lenses. All keratoconus patients wore corneal rigid gas permeable contact lenses (RGP) during the past 5 years. Control group subjects all wore soft contact lenses on a monthly wear daily basis during the past 3 years. Contact lens wearers in both groups used a multipurpose solution to maintain their contact lenses (RGP or soft contact lenses). All study participants were told not to wear their contact lenses for least 3 d prior to testing. Wearing time during the day and how many days they were without lenses were recorded. Objective testing included the Schirmer 1 test (without anesthetic),24 TBUT,25 corneal staining,26 Ap4A analysis,21,22,27 and impression cytology.20 Subjective testing involved recording subjective symptoms during questioning as well as grading dryness and discomfort using the ocular surface disease index OSDI questionnaire.28–30 Tear collections were always performed according to Van Bijsterveld criteria.24 A Schirmer strip (Tear Flo, HUB Pharmaceuticals, Rancho Cucamonga, CA) was Current Eye Research
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Keratoconus and dry eye 3
FIGURE 1 3D impression cytology of a KC patient. (A) Frontal section of two goblet cells showing diffuse layer of mucin (red in online version) on conjunctival epithelium. (B) Perpendicular section through frontal section shown in (A), used to evaluate goblet cell layer thickness (CLT) and mucin cloud height (MCH).
placed on the temporal tarsal conjunctiva of the lower lid for 5 min with the eyes closed. Tears volume was recorded as mm of moistened strip, followed by the Schirmer strips being placed in Eppendorf tubes containing 500 ml of ultrapure water. Samples were then frozen until high-pressure liquid chromatography (HPLC) analysis was performed. After the Schirmer I test, fluorescein was applied to evaluate TBUT and corneal staining. In order to assure repeatability of the staining procedure, a solution was prepared using a 10% concentration of sodium fluorescein diluted in saline (NaCl 0.9%). For each application, a micropipette with 5 ml of diluted fluorescein solution was applied to the inferior conjunctival sac. Then, 20 s later, TBUT was analyzed with a chronograph after the patient was asked to blink twice and keep their eyes open. The cornea was divided into five areas to record the grade of staining, as described by the Report of National Eye Institute, Industry-Sponsored Dry Eye Workshop26 and Cornea and Contact Lens Research Unit (CCLRU). All subjects completed the OSDI questionnaire. This questionnaire, which has been evaluated by several studies,28–30 comprises 12 questions, each with five possible responses designed to (i) grade the degree of DES symptoms and (ii) differentiate DES patients from healthy patients. OSDI scoring is as follows: 0 = anytime, 1 = sometimes, 2 = half the time, 3 = most of the time, and 4 = all of the time. Total scores range from 0 to 100, where 100 represents the highest degree of DES symptomatology. For Ap4A analysis, Schirmer I test strips were collected and placed in Eppendorf tubes containing 500 ml of ultrapure water, vortexed for 5 min, and carefully rinsed. The remaining liquid was placed in a 100 C bath for 20 min to precipitate proteins. Tubes !
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were then centrifuged at 4000 rpm for 30 min to form protein pellets. Quantification determination of Ap4A was performed by high-performance liquid chromatography (HPLC). Supernatants were chromatographed using SEP-PAK Accell QMA cartridges (Waters, Milford, MA). In brief, 250 ml of supernatant was passed through the cartridges, which had been equilibrated with 3 ml of ultrapure water. Elution of nucleotides and dinucleotides was performed by applying 1 ml of a solution containing 0.2 M KCl and 0.1 M HCl, after which samples were neutralized with KOH. The resulting eluents were then made up to a volume of 10:100 ml and analyzed by HPLC. We followed the same protocol described in previous studies.21,22,27 For impression cytology, we used an EyePrim (EyePrim,TMOPIA Technologies SAS, Paris, France), equipped with a polyethersulfone (PES) membrane. This device was placed in contact with an area 1.5– 2.0 mm from the corneal limbus, on the temporal portion of the bulbar conjunctiva, using gentle pressure for 2 s. Using this technique, it was possible to collect tissue without the need to anesthetize the conjunctiva. These impression cytology samples were preserved in 96% ethanol, processed with periodic acid Shiff (PAS) reagent, dehydrated through an ethanol series to xylol, and mounted on coverslips for microscopic observation. Microscopic observation was accomplished using a laser scanning confocal microscope (LCM) (Zeiss LSM Pascal; Carl Zeiss, Jena, Germany), following the protocol described by Peral and Pintor.20 Samples were viewed at magnifications of 20, 40, and 100 (oil immersion). This LCM is able to scan samples on the Z-axis at 1-mm intervals between photograms, and was used to determine CLT, MCH, and cell density, following a procedure described elsewhere20 (Figure 1).
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G. Carracedo et al. TABLE 2 Comparison of parameters between keratoconus and control groups. Variable Schirmer I test (mm) TBUT (s) Corneal staining (score) OSDI (score) Ap4A (mM) Goblet cell density (cells/mm2) CLT (mm) MCH (mm)
Keratoconus group 5.70 8.70 3.30 44.96 2.56 84.88 5.13 6.77
(3.74) (4.22) (1.70) (8.65) (1.10) (32.08) (0.99) (2.51)
Control group 12.44 9.94 1.44 17.78 0.15 128.88 8.31 8.89
(8.83) (6.26) (1.13) (6.50) (0.12) (50.6) (2.01) (2.98)
p Value 0.042 0.610 0.01 50.001 50.001 0.030 50.001 0.044
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p50.05; Wilcoxon Rank Sum test.
FIGURE 2 Comparison of mucin cloud height (MCH) and cell layer thickness (CLT) in KC and control groups. Photomicrograph shows that MCH and CLT are significantly decreased in the KC group. Data show mean ± SD. *p50.05; U Mann–Whitney test.
Statistical analyses were performed using version 15.0 SPSS software (SPSS Inc., Chicago, IL). Results are expressed as mean ± standard deviation (SD). Normality of samples was analyzed using the Shapiro-Wilk test, followed by the non-parametric Wilcoxon Rank Sum test to compare groups. Statistical significance was set as p50.05.
RESULTS Of the KC patients diagnosed in the study, 10 (66.67%) were grade II and five (33.33%) were grade III, according to the KC severity score grading scale.31 No differences were found between groups for other tests (Table 2). Contact lens wearers reported wearing their lenses 10 h/d, but did not wear their contact lenses for at least 3 d prior to testing. Schirmer testing indicated that the control group had higher tear
volumes than did the KC group (p50.05). TBUT was shorter for KC group patients than for control group subjects, but the differences were not statistically significant. Corneal staining results were statistically significant, with corneal staining scores 100% higher in KC group patients than in control subjects (p50.05). KC subjects had significantly higher overall scores on the OSDI questionnaire than did control subjects, indicating a more severe dry eye symptomatology (p50.001). KC patients mainly reported light sensitivity, sore eyes, and blurred vision. Moreover, KC patients’ eyes were affected uncomfortably under windy conditions. Control group subjects with the highest scores were those who reported sore eyes and light sensitivity. The 3D method of observation, which has been used to generate measurements of MCH, CLT, and cell density,20 showed that KC patients had a reduced Current Eye Research
Keratoconus and dry eye 5 MCH compared with control group subjects (p50.05) (Figure 2). For CLT, there was a statistically significant difference between groups (p50.001). KC patients showed a greater reduction in CLT compared with control subjects. As for goblet cell density, there was a statistically significant difference between groups (p = 0.03), with KC patients having fewer goblet cells (Figure 2). Finally, tear concentrations of Ap4A were significantly increased in the KC group compared with the control group (2.56 ± 1.10 and 0.15 ± 0.12 mM; p50.05).
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DISCUSSION Very few studies have correlated KC with DES. The most thorough study is that of Dogru et al.,3 who studied tear function and ocular surface changes in KC. Recent KC tear studies have focused on proinflammatory molecules and their role in ectasia progression.15,32–34 The Dry Eye Workshop (2007) designated ocular surface inflammation as being a possible sign of DES. This conclusion led us to consider the existence of a close relationship between DES and KC. Our study is the first to (i) correlate Ap4A concentrations and 3D impression cytology with symptoms of DES (using a validated questionnaire) and (ii) compare these parameters between KC patients and healthy subjects. In our study, KC patients showed significantly reduced tear volumes compared with control subjects. Our results were very close to the limiting values that indicate, according to the criterion of Van Bjisterveld,24 the existence of DES. Moreover, we found TBUT values to be reduced in 73% of KC group patients and, although they were not statistically significant, these results coincide with those of Dogru et al.3 One possible explanation for this is the irregular corneal surface, which would does not permit formation of an adequate tear film. Zemova et al.,35 however, found a relationship between dry eye parameters and corneal irregularity. Additional study is necessary to clarify this point. We found a significantly greater corneal staining in KC patients compared with control subjects. This might be related to DES, due either to the association of dry eye with corneal staining36 or to the tendency of KC patients to rub their eyes.14 OSDI is a well-reviewed and validated questionnaire28 for diagnosing DES. To our knowledge, ours is the first study to use this questionnaire to evaluate dry eye in KC patients. We found KC patients to have higher OSDI scores than control subjects. Dogru et al.3 indicated that 81.5% of their KC patients reported ocular discomfort, irritation, and foreign body sensations, all of which are indicative of ocular dryness. Our control group subjects showed mild scores for dry eye, most likely due to a response influenced by !
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discomfort at the end of the day, related to soft contact lenses. An important combination technique used in the present study was that of 3D conjunctival impression cytology for evaluation of mucin production by goblet cells. In 2008, Peral and Pintor20 revealed that evaluating cell density by impression cytology alone was inadequate, because some cells were not secreting mucin into the tears. Consequently, these workers proposed the use of impression cytology in combination with LCM for 3D analysis. Using this technique, it became possible to assess not only cell density but also MCH (which indicates cell secretion) and CLT (which indicates the amount of mucin contained within the goblet cells). We also used the EyePrimÕ ), a new device that not only permits tear collection without the need for a topical anesthetic but also provides reproducibility and efficacy. Our results showed a reduction in goblet cell density in KC patients, confirming results of several previous studies.3,37,38 Furthermore, KC goblet cells produced less mucin, i.e. a lower CLT, thus secreting less mucin into the tears, i.e. a lower MCH. These results explain the tear instability and high OSDI scores in KC group patients. The last characteristic examined in this study was the evaluation of Ap4A, a molecule belonging to the dinucleotide family, which has already been identified as an objective marker for DES. The concentrations of Ap4A in human tears increase concomitantly with dry eye.21 In our study, KC patients had Ap4A concentrations that were 20 times higher than those of healthy subjects, indicative of DES. There are two possibilities to account for the higher concentrations of Ap4A in the tears of KC patients. First, the increase in Ap4A may be due to mechanical stimulation of epithelial cells,39 resulting from eye rubbing, which can induce the release of Ap4A, as previously demonstrated in DES patients.20 Second, KC patients had greater degrees of corneal staining. The relationship between Ap4A and corneal wound healing has been established and is based on the ability of Ap4A in tears to accelerate the rate of healing, thus restoring corneal integrity.40 The difference in ages between groups could be considered a limiting factor, only if the mean age is taken into account. Signs and symptoms of dry eye worsen with age. Generally, the age of onset is 45–50 years and is related to hormonal factors.41 In our KC patients, however, there was no relationship between age and the parameters evaluated. Future studies with larger study populations may be necessary to evaluate the symptomatology of dry eye in KC patients as a function of age, including ages of up to 70 years. In contrast, perhaps due to the limited number of grade III KC patients, no differences in the severity KC were found and, thus, we cannot determine if KC severity correlates significantly with dry
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eye severity. Further studies are necessary to clarify this point. One limitation of this study might be the fact that KC patients had long histories of RGP contact lens wear, while most control subjects wore soft contact lenses. Lema et al.17 found that gas permeable contact lens-wear increases the levels of pro-inflammatory molecules in KC patient tears. Conversely, several studies have demonstrated that soft contact lens-wear provokes symptoms of dryness, especially at the end of the day.42,43 Moreover, no statistical differences were found either in impression cytology44 or tear film surface quality45 with the use of either type of contact lens materials. Despite these findings, however, we believe that the difference in contact lens types between groups is an important factor. Hence, further studies to assess the influence of contact lens wear on dry eye signs and symptoms in KC patients versus control subjects should be undertaken. In conclusion, the parameters evaluated in this study indicate that patients with KC suffer more severe symptoms of dry eye and tear instability than do non-KC patients, presumably due to decreased mucin levels in the tears of KC patients.
ACKNOWLEDGEMENTS The authors thank Michael J. Lipson for help in the preparation of the manuscript.
DECLARATION OF INTEREST The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper
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RESEARCH REPORT
Signs and Symptoms of Dry Eye in Keratoconus Patients: A Pilot Study Gonzalo Carracedo1, Alberto Recchioni1, Nicola´s Alejandre-Alba2, Alba Martin-Gil3, Almudena Crooke3, Ignacio Jimenez-Alfaro Morote2 and Jesu´s Pintor3
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1
Department of Optics II, Optometry and Vision, Faculty of Optics, Universidad Complutense de Madrid, Madrid, Spain, 2Department of Ophthalmology, Fundacio´n Jimenez Diaz, Madrid, Spain, and 3Department of Biochemistry and Molecular Biology IV, Faculty of Optics, Universidad Complutense de Madrid, Madrid, Spain
ABSTRACT Purpose: To compare signs and symptoms of dry eye in keratoconus (KC) patients versus healthy subjects. Methods: A total of 15 KC patients (KC group, n = 15 eyes) and 16 healthy subjects (control group, 16 eyes) were enrolled in this study. The Schirmer I test with no anesthetic, tear break-up time (TBUT), corneal staining characteristics, and ocular surface disease index (OSDI) scores were evaluated for both groups. Impression cytology, combined with/scanning laser confocal microscopy (LCM), was performed to evaluate goblet cell density, mucin cloud height (MCH), and goblet cell layer thickness (CLT). Finally, tear concentrations of di-adenosine tetraphosphate (Ap4A) were assessed. Results were statistically analyzed using Shapiro–Wilk and non-parametric Wilcoxon rank sum tests. Statistical significance was set at p50.05. Results: KC patients had lower tear volumes and greater corneal staining than did healthy subjects (p50.05). OSDI scores were 44.96 ± 8.65 and 17.78 ± 6.50 for the KC and control groups, respectively (p50.05). We found no statistically significant differences in TBUT between groups. Impression cytology revealed lower goblet cell densities in KC group patients versus control group subjects (84.88 ± 32.98 and 128.88 ± 50.60 cells/mm,2 respectively, p50.05). There was a statistically significant reduction in MCH and CLT in KC group patients compared with control group subjects. Ap4A tear concentrations were higher in KC group patients than in control group subjects (2.56 ± 1.10 and 0.15 ± 0.12 mM, respectively, p50.05). Conclusions: The parameters evaluated in this study indicate that KC patients suffer greater symptoms of dry eye and greater tear instability, primarily due to the decreased mucin production in their tears, than do healthy patients with no KC Keywords: 3D impression cytology, dinucleotide, dry eye, keratoconus
INTRODUCTION
presents between the first and the third decades of life, and is rarely congenital.6 The prevalence of the disease is 0.2–2.3% of the population.7–10 The multifactorial etiology of KC includes genetic factors,11 allergy,12 UV light exposure, and eye rubbing. Eye rubbing, which has been reported in 40–70% of keratoconus patients,13 is an event that is stimulated by ocular discomfort, i.e. burning, itching, and dry eyes.14 A recent clinical study demonstrated reduced tear quality, as evidenced by a reduced tear
Keratoconus (KC) is an ectatic disease of the central and paracentral cornea. The disease primarily not only involves Bowman’s membrane but also includes the anterior and middle stroma.1,2 Keratoconus creates alterations of the corneal surface that result in visual distortion, reduced tear film quality,3 and ocular discomfort.4 Keratoconus is a bilateral, progressive, and asymmetric disorder.5 It initially
Received 13 December 2013; revised 4 November 2014; accepted 9 November 2014; published online 9 December 2014 Correspondence: Gonzalo Carracedo, Department Optics II (Optometry and Vision), Faculty of Optics, Universidad Complutense de Madrid, C/Arcos del Jalon 118, 28032 Madrid, Spain. E-mail: [email protected]
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G. Carracedo et al. TABLE 1 Demographics. Parameter
Keratoconus group
Control group
p Value
No. eyes (no. patients) Mean age (years) ± SD Age range (years) Gender (male, female) Mean keratometry (D) This not mentioned in manuscript Flat Steep
15 (15) 39.30 ± 6.49 18–44 8, 7
16 (16) 26.56 ± 6.50 20–34 8, 8
– 50.001 – –
49.46 ± 6.32 56.35 ± 8.76
43.87 ± 2.53 45.31 ± 1.39
0.003 50.001
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p50.05, Wilcoxon Rank Sum test.
break-up time (TBUT), and increased corneal staining in KC patients.3 Biomicroscoy of KC patients who wear rigid gas permeable (RGP) contact lenses often reveals signs of mechanical trauma to the lens. This, in combination with frequent eye rubbing, typically leads to a strong inflammatory response – interleukin (IL) 1-a and -b – in the corneal epithelium. This inflammation, in turn, induces apoptosis of corneal stromal cells and fibroblasts.15–17 Because the ocular surface signs of KC are similar to those of dry eye syndrome (DES), we investigated the potential relationship between KC and dry eye. Impression cytology, considered the gold standard for diagnosing DES,18 assesses the density of the mucin-producing conjunctival goblet cells, which are critical for maintaining ocular surface integrity.19 A recently developed LCM technique for analyzing impression cytology allows 3D imaging of goblet cell density, CLT, and mucin cloud (MC) height.20 Using this combination technique, Peral and Pintor20 discerned that a number of goblet cells were not secreting mucins. Impression cytology/LCM thus provides more meaningful and objective details for diagnosis that previously available. Besides goblet cell analysis, testing for other substances present in tears can serve as markers of ocular surface dryness. One such component is di-adenosine tetraphosphate (Ap4A), which has been proposed as being a potential molecular biomarker for DES.21 In symptomatic, normal tear-producing, non-Sjo¨gren dry-eye patients, Ap4A levels were shown to be increased five-fold and were almost 100 times greater than in symptomatic non-Sjo¨gren dry-eye patients with low tear production. Additionally, Ap4A concentrations were increased to 42-fold in Sjo¨gren dry-eye patients compared with normal individuals.22 To the best of our knowledge, no previous studies have correlated symptoms of DES with objective measures, e.g., Ap4A concentrations or goblet cell3D analysis, in KC patients. Thus, the objective of this study was to evaluate if keratoconus patients have more signs and symptoms of DES than do nonkeratoconus patients.
MATERIALS AND METHODS This pilot, experimental, prospective study included 15 keratoconus subjects (eight men and seven women), mean ages of 39.30 ± 6.49 (range, 18–44) years old and 16 healthy subjects (eight men and eight women), mean ages of 26.56 ± 6.50 (range, 20–34) years, all of had been treated at the Fundacio´n Jime´nez-Dı´az, Madrid, Spain, and the University Clinic of Optometry, Complutense University, Madrid, Spain. Subjects already diagnosed with DES or subjects undergoing treatment for DES were excluded. All subjects took part voluntarily in the study and were free to leave without question if they chose. All subjects provided informed consent after, the nature of the research and the risks of participation were explained. The study adhered to the tenets of the Declaration of Helsinki23 and was approved by the Ethics Committee (CEIC) of the Fundacio´n Jime´nez-Dı´az. Detailed demographic characteristics of the study population are shown in Table 1. All subjects were contact lens wearers, except two control subjects who had never worn contact lenses. All keratoconus patients wore corneal rigid gas permeable contact lenses (RGP) during the past 5 years. Control group subjects all wore soft contact lenses on a monthly wear daily basis during the past 3 years. Contact lens wearers in both groups used a multipurpose solution to maintain their contact lenses (RGP or soft contact lenses). All study participants were told not to wear their contact lenses for least 3 d prior to testing. Wearing time during the day and how many days they were without lenses were recorded. Objective testing included the Schirmer 1 test (without anesthetic),24 TBUT,25 corneal staining,26 Ap4A analysis,21,22,27 and impression cytology.20 Subjective testing involved recording subjective symptoms during questioning as well as grading dryness and discomfort using the ocular surface disease index OSDI questionnaire.28–30 Tear collections were always performed according to Van Bijsterveld criteria.24 A Schirmer strip (Tear Flo, HUB Pharmaceuticals, Rancho Cucamonga, CA) was Current Eye Research
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Keratoconus and dry eye 3
FIGURE 1 3D impression cytology of a KC patient. (A) Frontal section of two goblet cells showing diffuse layer of mucin (red in online version) on conjunctival epithelium. (B) Perpendicular section through frontal section shown in (A), used to evaluate goblet cell layer thickness (CLT) and mucin cloud height (MCH).
placed on the temporal tarsal conjunctiva of the lower lid for 5 min with the eyes closed. Tears volume was recorded as mm of moistened strip, followed by the Schirmer strips being placed in Eppendorf tubes containing 500 ml of ultrapure water. Samples were then frozen until high-pressure liquid chromatography (HPLC) analysis was performed. After the Schirmer I test, fluorescein was applied to evaluate TBUT and corneal staining. In order to assure repeatability of the staining procedure, a solution was prepared using a 10% concentration of sodium fluorescein diluted in saline (NaCl 0.9%). For each application, a micropipette with 5 ml of diluted fluorescein solution was applied to the inferior conjunctival sac. Then, 20 s later, TBUT was analyzed with a chronograph after the patient was asked to blink twice and keep their eyes open. The cornea was divided into five areas to record the grade of staining, as described by the Report of National Eye Institute, Industry-Sponsored Dry Eye Workshop26 and Cornea and Contact Lens Research Unit (CCLRU). All subjects completed the OSDI questionnaire. This questionnaire, which has been evaluated by several studies,28–30 comprises 12 questions, each with five possible responses designed to (i) grade the degree of DES symptoms and (ii) differentiate DES patients from healthy patients. OSDI scoring is as follows: 0 = anytime, 1 = sometimes, 2 = half the time, 3 = most of the time, and 4 = all of the time. Total scores range from 0 to 100, where 100 represents the highest degree of DES symptomatology. For Ap4A analysis, Schirmer I test strips were collected and placed in Eppendorf tubes containing 500 ml of ultrapure water, vortexed for 5 min, and carefully rinsed. The remaining liquid was placed in a 100 C bath for 20 min to precipitate proteins. Tubes !
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were then centrifuged at 4000 rpm for 30 min to form protein pellets. Quantification determination of Ap4A was performed by high-performance liquid chromatography (HPLC). Supernatants were chromatographed using SEP-PAK Accell QMA cartridges (Waters, Milford, MA). In brief, 250 ml of supernatant was passed through the cartridges, which had been equilibrated with 3 ml of ultrapure water. Elution of nucleotides and dinucleotides was performed by applying 1 ml of a solution containing 0.2 M KCl and 0.1 M HCl, after which samples were neutralized with KOH. The resulting eluents were then made up to a volume of 10:100 ml and analyzed by HPLC. We followed the same protocol described in previous studies.21,22,27 For impression cytology, we used an EyePrim (EyePrim,TMOPIA Technologies SAS, Paris, France), equipped with a polyethersulfone (PES) membrane. This device was placed in contact with an area 1.5– 2.0 mm from the corneal limbus, on the temporal portion of the bulbar conjunctiva, using gentle pressure for 2 s. Using this technique, it was possible to collect tissue without the need to anesthetize the conjunctiva. These impression cytology samples were preserved in 96% ethanol, processed with periodic acid Shiff (PAS) reagent, dehydrated through an ethanol series to xylol, and mounted on coverslips for microscopic observation. Microscopic observation was accomplished using a laser scanning confocal microscope (LCM) (Zeiss LSM Pascal; Carl Zeiss, Jena, Germany), following the protocol described by Peral and Pintor.20 Samples were viewed at magnifications of 20, 40, and 100 (oil immersion). This LCM is able to scan samples on the Z-axis at 1-mm intervals between photograms, and was used to determine CLT, MCH, and cell density, following a procedure described elsewhere20 (Figure 1).
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G. Carracedo et al. TABLE 2 Comparison of parameters between keratoconus and control groups. Variable Schirmer I test (mm) TBUT (s) Corneal staining (score) OSDI (score) Ap4A (mM) Goblet cell density (cells/mm2) CLT (mm) MCH (mm)
Keratoconus group 5.70 8.70 3.30 44.96 2.56 84.88 5.13 6.77
(3.74) (4.22) (1.70) (8.65) (1.10) (32.08) (0.99) (2.51)
Control group 12.44 9.94 1.44 17.78 0.15 128.88 8.31 8.89
(8.83) (6.26) (1.13) (6.50) (0.12) (50.6) (2.01) (2.98)
p Value 0.042 0.610 0.01 50.001 50.001 0.030 50.001 0.044
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p50.05; Wilcoxon Rank Sum test.
FIGURE 2 Comparison of mucin cloud height (MCH) and cell layer thickness (CLT) in KC and control groups. Photomicrograph shows that MCH and CLT are significantly decreased in the KC group. Data show mean ± SD. *p50.05; U Mann–Whitney test.
Statistical analyses were performed using version 15.0 SPSS software (SPSS Inc., Chicago, IL). Results are expressed as mean ± standard deviation (SD). Normality of samples was analyzed using the Shapiro-Wilk test, followed by the non-parametric Wilcoxon Rank Sum test to compare groups. Statistical significance was set as p50.05.
RESULTS Of the KC patients diagnosed in the study, 10 (66.67%) were grade II and five (33.33%) were grade III, according to the KC severity score grading scale.31 No differences were found between groups for other tests (Table 2). Contact lens wearers reported wearing their lenses 10 h/d, but did not wear their contact lenses for at least 3 d prior to testing. Schirmer testing indicated that the control group had higher tear
volumes than did the KC group (p50.05). TBUT was shorter for KC group patients than for control group subjects, but the differences were not statistically significant. Corneal staining results were statistically significant, with corneal staining scores 100% higher in KC group patients than in control subjects (p50.05). KC subjects had significantly higher overall scores on the OSDI questionnaire than did control subjects, indicating a more severe dry eye symptomatology (p50.001). KC patients mainly reported light sensitivity, sore eyes, and blurred vision. Moreover, KC patients’ eyes were affected uncomfortably under windy conditions. Control group subjects with the highest scores were those who reported sore eyes and light sensitivity. The 3D method of observation, which has been used to generate measurements of MCH, CLT, and cell density,20 showed that KC patients had a reduced Current Eye Research
Keratoconus and dry eye 5 MCH compared with control group subjects (p50.05) (Figure 2). For CLT, there was a statistically significant difference between groups (p50.001). KC patients showed a greater reduction in CLT compared with control subjects. As for goblet cell density, there was a statistically significant difference between groups (p = 0.03), with KC patients having fewer goblet cells (Figure 2). Finally, tear concentrations of Ap4A were significantly increased in the KC group compared with the control group (2.56 ± 1.10 and 0.15 ± 0.12 mM; p50.05).
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DISCUSSION Very few studies have correlated KC with DES. The most thorough study is that of Dogru et al.,3 who studied tear function and ocular surface changes in KC. Recent KC tear studies have focused on proinflammatory molecules and their role in ectasia progression.15,32–34 The Dry Eye Workshop (2007) designated ocular surface inflammation as being a possible sign of DES. This conclusion led us to consider the existence of a close relationship between DES and KC. Our study is the first to (i) correlate Ap4A concentrations and 3D impression cytology with symptoms of DES (using a validated questionnaire) and (ii) compare these parameters between KC patients and healthy subjects. In our study, KC patients showed significantly reduced tear volumes compared with control subjects. Our results were very close to the limiting values that indicate, according to the criterion of Van Bjisterveld,24 the existence of DES. Moreover, we found TBUT values to be reduced in 73% of KC group patients and, although they were not statistically significant, these results coincide with those of Dogru et al.3 One possible explanation for this is the irregular corneal surface, which would does not permit formation of an adequate tear film. Zemova et al.,35 however, found a relationship between dry eye parameters and corneal irregularity. Additional study is necessary to clarify this point. We found a significantly greater corneal staining in KC patients compared with control subjects. This might be related to DES, due either to the association of dry eye with corneal staining36 or to the tendency of KC patients to rub their eyes.14 OSDI is a well-reviewed and validated questionnaire28 for diagnosing DES. To our knowledge, ours is the first study to use this questionnaire to evaluate dry eye in KC patients. We found KC patients to have higher OSDI scores than control subjects. Dogru et al.3 indicated that 81.5% of their KC patients reported ocular discomfort, irritation, and foreign body sensations, all of which are indicative of ocular dryness. Our control group subjects showed mild scores for dry eye, most likely due to a response influenced by !
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discomfort at the end of the day, related to soft contact lenses. An important combination technique used in the present study was that of 3D conjunctival impression cytology for evaluation of mucin production by goblet cells. In 2008, Peral and Pintor20 revealed that evaluating cell density by impression cytology alone was inadequate, because some cells were not secreting mucin into the tears. Consequently, these workers proposed the use of impression cytology in combination with LCM for 3D analysis. Using this technique, it became possible to assess not only cell density but also MCH (which indicates cell secretion) and CLT (which indicates the amount of mucin contained within the goblet cells). We also used the EyePrimÕ ), a new device that not only permits tear collection without the need for a topical anesthetic but also provides reproducibility and efficacy. Our results showed a reduction in goblet cell density in KC patients, confirming results of several previous studies.3,37,38 Furthermore, KC goblet cells produced less mucin, i.e. a lower CLT, thus secreting less mucin into the tears, i.e. a lower MCH. These results explain the tear instability and high OSDI scores in KC group patients. The last characteristic examined in this study was the evaluation of Ap4A, a molecule belonging to the dinucleotide family, which has already been identified as an objective marker for DES. The concentrations of Ap4A in human tears increase concomitantly with dry eye.21 In our study, KC patients had Ap4A concentrations that were 20 times higher than those of healthy subjects, indicative of DES. There are two possibilities to account for the higher concentrations of Ap4A in the tears of KC patients. First, the increase in Ap4A may be due to mechanical stimulation of epithelial cells,39 resulting from eye rubbing, which can induce the release of Ap4A, as previously demonstrated in DES patients.20 Second, KC patients had greater degrees of corneal staining. The relationship between Ap4A and corneal wound healing has been established and is based on the ability of Ap4A in tears to accelerate the rate of healing, thus restoring corneal integrity.40 The difference in ages between groups could be considered a limiting factor, only if the mean age is taken into account. Signs and symptoms of dry eye worsen with age. Generally, the age of onset is 45–50 years and is related to hormonal factors.41 In our KC patients, however, there was no relationship between age and the parameters evaluated. Future studies with larger study populations may be necessary to evaluate the symptomatology of dry eye in KC patients as a function of age, including ages of up to 70 years. In contrast, perhaps due to the limited number of grade III KC patients, no differences in the severity KC were found and, thus, we cannot determine if KC severity correlates significantly with dry
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eye severity. Further studies are necessary to clarify this point. One limitation of this study might be the fact that KC patients had long histories of RGP contact lens wear, while most control subjects wore soft contact lenses. Lema et al.17 found that gas permeable contact lens-wear increases the levels of pro-inflammatory molecules in KC patient tears. Conversely, several studies have demonstrated that soft contact lens-wear provokes symptoms of dryness, especially at the end of the day.42,43 Moreover, no statistical differences were found either in impression cytology44 or tear film surface quality45 with the use of either type of contact lens materials. Despite these findings, however, we believe that the difference in contact lens types between groups is an important factor. Hence, further studies to assess the influence of contact lens wear on dry eye signs and symptoms in KC patients versus control subjects should be undertaken. In conclusion, the parameters evaluated in this study indicate that patients with KC suffer more severe symptoms of dry eye and tear instability than do non-KC patients, presumably due to decreased mucin levels in the tears of KC patients.
ACKNOWLEDGEMENTS The authors thank Michael J. Lipson for help in the preparation of the manuscript.
DECLARATION OF INTEREST The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper
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