Diadenosine polyphosphates after laser in situ keratomileusis and photorefractive keratectomy refractive techniques

Diadenosine polyphosphates after laser in situ keratomileusis and photorefractive keratectomy refractive techniques Gonzalo Carracedo, 1 Isabel Cacho, 2 Juan Sanchez-Naves 2 and Jesus Pintor 3 1 Department of Optics II (Optometry and Vision), Faculty of Optics, Universidad Complutense de Madrid, Madrid, Spain 2 Instituto Balear de Oftalmologı a, Palma de Mallorca, Spain 3 Department of Biochemistry and Molecular Biology IV, Faculty of Optics, Universidad Complutense de Madrid, Madrid, Spain ABSTRACT. Purpose: To study the concentrations of diadenosine polyphosphates in the ocular surface after and. Methods: Sixty-one patients (30 males and 31 females) with ages ranging from 20 to 63 (34.04 ± 9.13 years) were recruited in Balear Institute of Ophthalmology, Palma de Mallorca, Spain. was performed in 92 eyes of 46 patients and in 25 eyes of 15 patients. Variations in the levels of diadenosine polyphosphate (Ap 4 A and Ap 5 A), Schirmer I (Jones test), TBUT, corneal staining together with the Dry Eye Questionnaire to evaluate discomfort and dryness were studied. All tests were performed at the preoperative visit and at 1-day, 2-week, 1-month and 3-month postoperative visits. Results: Ap 4 A showed a 5 and 3.5 fold increase at the 1-day visit for and, respectively. patients continued having higher statistically significant concentrations (p = 0.01) all over the follow-up. Ap 5 A showed no significant differences at any visit. Tear volume decreased during the 3 months in. The cases had a normal volume at 1 month. TBUT in increased at the 1-day visit (p = 0,002) and decreased from the 2 weeks onwards and for the, decreased by a 35% at the 1-day visit and kept reduced for a month. Discomfort only increased at the 1-day visit (p = 0.007). Dryness frequency was similar in all visits. Conclusions: Ap 4 A levels only are increased in refractive surgery patients during the first day after the surgery. This increasing suggests that Ap 4 A may help accelerating the healing process. Key words: dinucleotides dry eye wound healing Acta Ophthalmol. 2014: 92: e5 e11 ª 2013 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd. doi: 10.1111/aos.12194 Introduction Laser in situ keratomileusis () and photorefractive keratectomy () are safe and effective refractive surgery methods producing similar refractive outcomes. Both techniques produce changes in ocular surface increasing dry eye sensation (Nejima et al. 2005; Toda 2008). In, debridement of corneal epithelium and stromal ablation damages the corneal sensory nerves. On the other hand, in, the lamellar flat cut induces sensory deprivation by severing deeper stromal nerve. This nerve ablation strongly suggests several ocular surface problems including tear function (Perez-Santonja et al. 1999). Several studies relate differences depending on refractive surgery technique used regarding the associated dry eye. It seems that produces stronger eye dryness than. Patients submitted to refractive surgery present a decrease in tear secretion, blink rate, corneal and conjunctival epithelial integrity and conjunctival goblet cell density when compared with normal individuals. These alterations decrease tear film stability and may lead to dry eye symptoms during the first year after surgery (Benitez-del-Castillo et al. 2001; Rodriguez-Prats et al. 2007). The Dry Eye Workshop proposed to diagnose dry eye combining the use of a validated questionnaire regarding symptoms, a test to evaluate ocular surface damage, the measurement of tear instability and tear secretion, and tear hyperosmolarity (listed] 2007). However, it is necessary to be aware that the diagnosis of dry eye is difficult due to the poor correlation between dry eye signs and symptoms (Nichols et al. 2004). Analytical techniques are providing valuable information about new e5

components in tears that may have important biochemical and physiological functions (Peral et al. 2008). Among these new tear components, diadenosine polyphosphate emerge as interesting compounds due to their intracellular and extracellular physiological actions. In the tear film, it is known that diadenosine tetraphosphate (Ap 4 A) and diadenosine pentaphosphate (Ap 5 A) concentrations rise in patients with dry eye symptoms and Sjo gren s syndrome, suggesting the possibility that these compounds could be objective markers to score dry eye (Peral et al. 2006; Carracedo et al. 2010). Moreover, a single-dose topical application of the dinucleotide Ap 4 Ain rabbits can stimulate tear secretion, giving this compound a secretagogue action (Pintor et al. 2002a,b). Another role of this dinucleotide on the ocular surface Ap 4 A is improving the rate of corneal wound healing in New Zealand white rabbits (Pintor et al. 2004). Due to the involvement of diadenosine polyphosphates in the ocular surface physiopathology, it is a matter of interest to investigate a possible relationship of this substance in refractive surgery patients. With this aim, the present experimental work describes variations in the levels of diadenosine polyphosphate (Ap 4 A and Ap 5 A), during 3 months after and. Furthermore, we examined tear secretion and its stability, corneal staining and discomfort and dryness symptoms, which may be related during dry eye. Methods The study was conducted in compliance with good clinical practice guidelines, informed consent regulations and the tenets of the Declaration of Helsinki (WMA, 2008). The study was approved by the Balear Institute of Ophthalmology IRB. All of the subjects enrolled in the study were adults older than 18 years who were able to give informed consent. Patients Sixty-one patients (30 men and 31 women) with ages ranging from 20 to 63 (average 34.04 ± 9.13 years) were recruited. was performed in 92 eyes of 46 patients and in 25 eyes of 15 patients. More detailed demographic characteristics of the population being sampled are shown in Table 1. We followed the inclusion and exclusion criteria for dry eye and did not include any cases or severe dry eye or Sjo gren s syndrome at the preoperative visit. Patients were subjected to a complete preoperative ophthalmic examination including the tests for this study. Postoperative visits were scheduled at 1 day, 2 weeks, 1 and 3 months after surgery. Surgery procedures All surgeries were performed by the same surgeon (J.S.N) using the Technolas 217 flying spot excimer laser system, version V 312.383 (BAUSCH & LOMB, Irvine, CA, USA). Laser parameters included the following: wavelength of 193 nm, radiant exposure (Fluence) of 160 mj cm 2, pulse repetition rate of 50 Hz, average ablation depth pulse of 0.25 lm on the cornea and an ablation zone diameter from 6.5 to 7 mm with a transition zone of 0.5 mm. For all eyes, preoperative manifest refraction was selected as the target correction. For cases, the XP automated microkeratome (BAUSCH & LOMB, Irvine, CA, USA) was used to create a superior-hinged corneal flap (120 or 140 lm head plates). These patients were prescribed topical antibiotic and steroid drops (Tobradex, Alcon, Fort Worth, TX, USA) every 6 hr for 5 days. Artificial tears (Oculotec, Novartis, Basel, Switzerland) were also prescribed a minimum of four times per day. In cases of severe symptoms signs, patients were changed to other artificial tears containing hyaluronic acid (Hyaback, Thea, Barcelone, Spain). Artificial tears were prescribed for a minimum of 6 weeks but as long as needed. The technique was only applied to patients who had up to a maximum of )3.00 dioptres. Only BSS solution was used to soften the epithelial cell layer and then a blade to scrape and remove it. After, Tobradex drops were prescribed and tapered during a month (reducing from 4 times day during the first week to 3, 2 and finally 1 time day during the fourth week). Artificial tears were prescribed following the same criteria than that used for patients. Finally, a bandage contact lens was applied straight after the surgery and worn for 5 days. Trials Tear secretion was measured using the Schirmer I test (Jones test) with anaesthesia. The tear collection was always performed following van Bijsterveld criteria (van Bijsterveld 1969). The Schirmer strip (Tear Flo, HUB pharmaceuticals, Rancho Cucamonga, CA, USA) was placed on the temporal tarsal conjunctiva of the lower lid for 5 min with the eyes closed. The volume of tears, as millimetres of moistened strip, was recorded and the Schirmer strips were placed in Eppendorf tubes containing 500 ll of ultrapure water, then the samples were frozen until the high-pressure liquid chromatography (HPLC) analysis was performed (Peral et al. 2006). After Schirmer I test (Jones test) with anaesthesia, fluorescein was applied to evaluate TBUT and corneal staining. In order to warrant 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 ll of diluted fluorescein solution was applied in the inferior conjunctival sac, and 20 seconds later, TBUT was analysed using a chronograph to record the time to break after the patient was asked to blink twice and remain with the eyes open. The cornea Table 1. Demographic information of the population enrolled in the study. Parameter Total Number of eyes (patients) 117 (61) 92 (46) 25 (15) Mean age (years) ± SD 34.55 ± 9.34 34.53 ± 9.24 34.64 ± 9.85 Age range (years) [20, 63] [20, 61] [24, 63] Gender (male female) [30, 31] [22, 26] [8, 5] Mean spherical equivalent (D) ± SD )2.06 ± 2.42 )2.21 ± 2.6 )1.35 ± 0.88 Range of spherical equivalent (D) [)6.38, 5.63] [)6.38, 5.63] [)2.50, 0.88] e6

was divided in five areas to record the grading staining as proposed the Report of the National Eye Institute and Industry-Sponsored Dry Eye Workshop and the Cornea and Contact Lens Research Unit (CCLRU) grading scales was used. We asked the patients to quantify typical symptoms of dry eye: discomfort and dryness. For this, they had to assess the frequency from 0 to 5, being five the maximum. These questions were taken from Dry Eye Questionnaire (DEQ) performed by Begley et al. (2001). Tear processing and HPLC analysis After thawing, the samples were strongly vortexed for 5 min. The strips were carefully rinsed and the liquid in the Eppendorf tube was heated in a 100 C bath for 20 min to precipitate proteins. In order to pellet the proteins, the tubes were centrifuged at 3500 g for 30 min. Diadenosine polyphosphates are not degraded by this treatment as previously demonstrated (15). Supernatants were chromatographed through SEP-PAK Accell QMA cartridges (16). Briefly, 250 ll of the supernatant was passed through the cartridges that were previously equilibrated with 3 ml of ultrapure water. The elution of the nucleotides and dinucleotides was performed by applying 1 ml of a solution containing 0.2 m KCl, 0.1 m HCl. Prior to injection into the HPLC, samples were neutralized with KOH. This eluents were injected at a volume of 10 100 ll. Determination and quantification of diadenosine polyphosphates were performed by HPLC. The chromatographic system consisted of a Waters 1515 isocratic HPLC pump, a 2487 dual absorbance detector and a Reodyne injector, all managed by the Breeze software from Waters. The column was a Novapack C18 (15 cm length, 0.4 cm diameter) from Waters. The system was equilibrated overnight with the following mobile phase: 0.1 m KH 2 PO 4,2mm tetrabutyl ammonium, 17% acetonitrile, ph 7.5 (12). Detection was monitored at 260 nm wavelength. All the peaks identified as putative dinucleotides were taken for phosphodiesterase treatment. Phosphodiesterase from Crotalus adamanteus, from Sigma (St. Louis, MO, USA; EC 3.1.4.1.) at a concentration of 1.0 U ml, was incubated at room temperature for 30 min with the corresponding putative dinucleotide. The digestion products were analysed by HPLC. Peaks were transformed into concentrations by means of external standards of known concentrations of diadenosine polyphosphates (Pintor et al. 2002a,b). Statistical analysis Data were analysed by statistical package SPSS, version 15.0 for Windows (SPSS, Inc., Chicago, IL, USA). Sample size calculations were performed with statistical software (Granmo 6.0; Institut Municipal d Investigacio n Medica, Barcelona, Spain). With an accepted two-sided statistical significance threshold of 0.05 and a risk of 0.20, for a standard deviation 0.7 units to the mean and in order to detect a difference of 0.6 units or more, 14 subjects were needed to find statistically significant differences. The values presented are the means ± SEM of the experiments performed. We selected randomly one eye per subject. Normality of distribution was assessed using the Kolgomorov Smirnov test. Parametric test was used to compare the studied groups. Differences between and groups in tears functions test were estimated by the Student s t-test for independent samples. We used the Student s t-test for related samples to assess the differences among different follow-up visits into each group. p < 0.05 was considered statistically significant. Ap4A concentration (μm) 0.800 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 Results Diadenosine polyphosphates Ap 4 A increases its concentration in tears in both and surgery in the next day after surgery, being increased 5 and 3.5 fold compared with the preoperative visit, respectively. After 1 month, Ap 4 A continues its increased concentration in patients with statistically significant p value (p = 0.007). For patients, Ap 4 A concentrations returned to baseline levels after 2 weeks, although the samples collected 1 month after the surgery, Ap 4 A showed lower concentrations than before surgery. The significant difference found between and patients in Ap 4 A concentration was obtained 1 month after surgery (p < 0.05). In the case of Ap 5 A, it remained stable during the 3 months of study. (Figs 1 and 2, Table 2). Tear schirmer For patients, tear volume was lower from the day after surgery and so on, being statistically significant after 2 weeks (p < 0.001), with a reduction by 30%. At 3 months, the tear volume decreased around 20% compared with preoperative values (p = 0.01). On the other hand, in surgery, tear volume at first day was 65% higher than the preoperative (p < 0.001), returning to normal values 1 month after the intervention. We found statistically significant differences between and patients during the first 2 weeks (Fig. 3, Table 2). Fig. 1. Concentrations of Ap 4 A during 3-month follow-up, both (46 patients; 92 eyes) and (15 patients; 25 eyes) surgeries. p value <0.05 versus. t-student test. e7

Ap5A concentration (μm) 0.200 0.180 0.160 0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000 Tear break-up time Fig. 2. Concentrations of Ap 5 A during 3-month follow-up, both (46 patients; 92 eyes) and (15 patients; 25 eyes) surgeries. t-student test. Table 2. Comparisons between baseline and different follow-up visits in and patients Test Visit p Value p value Ap 4 A mean (SEM) Pre-operative 0.095 (0.027) 0.128 (0.036) 1 day 0.508 (0.199) 0.005 0.453 (0.105) 0.048 2 weeks 0.231 (0.097) 0.009 0.135 (0.015) 0.531 1 month 0.245 (0.109) 0.007 0.068 (0.006) 0.157 3 months 0.159 (0.076) 0.245 0.151 (0.019) 0.912 Ap 5 A mean (SEM) Pre-operative 0.077 (0.026) 0.119 (0.054) 1 day 0.049 (0,026) 0.963 0.081 (0.026) 0.251 2 weeks 0.031 (0.008) 0.017 0.045 (0.017) 0.029 1 month 0.085 (0.003) 0.241 3 months 0.081 (0.027) 0.334 Schirmer (mm) mean (SEM) Pre-operative 20.08 (2.76) 19.05 (1.69) 1 day 18.11 (2.66) 0.367 29.87 (0.81) <0.001 2 weeks 13.89 (3,11) <0.001 22.56 (1.41) 0.024 1 month 16.04 (3.34) 0.012 16.84 (1.38) 0.967 3 months 15.35 (2.66) 0.01 19.57 (1.36) 0.945 BUT mean (SEM) Pre-operative 12.24 (0.64) 16.32 (1.67) 1 day 14.98 (2.11) 0.011 10.45 (0.67) 0.039 2 weeks 13.23 (2,65) 0.189 11.89 (0.80) 0.086 1 month 12.11 (1.79) 0.459 13.35 (1.13) 0.236 3 months 11.01 (1.81) 0.423 14.21 (1.07) 0.451 Corneal staining mean (SEM) Discomfort mean (SEM) Dryness mean (SEM) Pre-operative 0.52 (0.22) 0.57 (0.07) 1 day 1.03 (0.20) 0.043 0.89 (0.1) 0.007 2 weeks 1.07 (0.23) 0.006 1.03 (0.1) 0.011 1 month 0.65 (0.19) 0.388 0.54 (0.09) 0.645 3 months 0.49 (0.17) 0.889 0.54 (0.11) 0.666 Pre-operative 1.43 (0.14) 1.46 (0.33) 1 day 1.93 (0.12) 0.005 1.63 (0.26) 0.697 2 weeks 1.50 (0.10) 0.667 1.13 (0.17) 0.371 1 month 1.35 (0.09) 0.632 1.43 (0.20) 0.974 3 months 1.08 (0.08) 0.022 0.75 (0.18) 0.065 Pre-operative 1.21 (0.13) 1.31 (0.29) 1 day 1.47 (0.12) 0.161 1.25 (0.14) 0.876 2 weeks 1.64 (0.09) 0.01 1.5 (0.21) 0.571 1 month 1.24 (0.07) 0.895 1.14 (0.21) 0.655 3 months 1.24 (0.07) 0.895 1.00 (0.22) 0.404 p < 0.05. p < 0.01. Comparison post-operative with pre-operative values. In the case of, TBUT increased slightly next day (p = 0.011), but 2 weeks later, no significant differences with preoperative values were observed. At 3 months, patients showed TBUT values lower than preoperative visit, although it was not statistically significant. TBUT values decreased around 35% the day after surgery. One month after surgery still remained not statistically reduced in tear break-up time. Figure 4 shows a downward trend in patients, whereas in the, TBUT tends to return to initial values. Significant differences have been shown between the two surgeries in the preoperative, next day and 3 months after the surgery (Table 2). Corneal staining Corneal staining was similar in both surgeries, the first 2 weeks being significantly increased (p < 0.05) and returning to initial values 1 month after the surgery (Table 2). Symptoms We evaluated the frequency of two typical dry eye symptoms such as discomfort and dryness. In the case of discomfort, it increased the day after surgery (for both and ), being significant in (p = 0.005) but returning to preoperative values 2 weeks after the surgery. Three months after the surgical procedures, the discomfort frequency decreased, being statistically significant for (p = 0.022). Dryness frequency was similar after surgery than that of preoperative data in both surgical procedures. However, it was statistically significant in at 2 weeks (Table 2). Discussion Nucleotides and dinucleotides are important tear molecules in normal and pathological conditions (Peral et al. 2006; Guzman-Aranguez et al. 2007; Carracedo et al. 2010). The possible changes of these substances on patients submitted to refractive surgery have not yet been investigated. With this aim, we have evaluated the dinucleotide concentrations before and along 3-month postrefractive surgery follows up. Patients selected for this study were operated using two different techniques: and. Independently of the way the surgery has been performed, there was an increase in Ap 4 A levels. Ap 5 A concentration remained stable, with values e8

Tear secretion (mm) 40 35 30 25 20 15 10 5 0 Fig. 3. Schirmer test with anaesthesia during 3-month follow-up, both (46 patients; 92 eyes) and (15 patients; 25 eyes) surgeries. t-student test. Tear break up time (seconds) 30 25 20 15 10 5 0 Fig. 4. Tear break-up time (TBUT) during 3-month follow-up, both (46 patients; 92 eyes) and (15 patients; 25 eyes) surgeries. p value <0.05 versus. t-student test. lower than those measured at the preoperative stage. As indicated in other studies, Ap 4 A has the ability to accelerate corneal epithelial cell migration. In contrast, a high concentration of Ap 5 A performs the opposite effect of decreasing cell migration (Mediero et al. 2008). Moreover, we have measured Ap 4 A and Ap 5 A concentrations after intraocular collamer lens (ICL) surgery where the damage in the corneal epithelium is minimal. Both dinucleotides levels had a slightly increasing, but it was not statistically significant the day after the surgical procedure (unpublished data). Altogether, these results strongly suggest that the high concentrations of Ap 4 A found the next day of the surgery may help accelerating the healing process as described in animal models (Guzman-Aranguez et al. 2007). Patients undergoing showed elevated concentrations of Ap 4 A during the 3-month follow-up of the study (compared with the preoperative), although it was only statistically significant 1 month after the surgery. Ap 4 A concentration was roughly 2 3- folds lower than that found in patients with symptomatic dry eye and normal lacrimation or patients with Sjo gren s syndrome and normal lacrimation (Carracedo et al. 2010). The diadenosine polyphosphates Ap 4 A and Ap 5 A are released from corneal epithelium due to the shear force produced by the movement of the upper eyelid during the blinking process. The rise in the levels of both diadenosine polyphosphates is concomitant with the increase in the blinking frequency (Pintor et al. 2002a,b) and therefore, the decrease of corneal sensitivity after the surgery could be a factor affecting the release of the Ap 4 A and Ap 5 A (Collins et al. 1989). The tear volume for these patients decreased from the first day after surgery, being statistical significant until 2 weeks. These results are consistent with others that measure tear volume (Siganos et al. 2002) and are linked to a decrease in corneal sensitivity caused by the damage of corneal sensory nerve fibres (Patel et al. 2001; Belmonte et al. 2004). Concerning, these patients showed Ap 4 A concentrations similar to preoperative values from 2 weeks after the surgery. Tear volume in patients increased significantly the first day after surgery but returned to baseline in 1 month, and these results were similar to those found by other authors (Siganos et al. 2000; Lee et al. 2002). The increase of Ap 4 A the day after the surgery could be caused by the corneal epithelial debridement. It would be reasonable to think that higher concentrations of these dinucleotides should be expected, but it is probable that the use of the contact lens affects the dinucleotides concentration, reducing their levels because the shear stress due to the blinking should be less intense. In addition to dinucleotide concentrations and tear secretion, we also assessed TBUT and corneal staining. We observed no significant differences in TBUT in patients during the 3 month of study. Some authors report a reduction in TBUT in and patients (Toda et al. 2001; Konomi et al. 2008), whereas others found no significant differences in or in (Nejima et al. 2005; Mian et al. 2009). Our findings of increased corneal staining during the first 2 weeks in and match with previous findings described by other authors (Toda et al. 2001). There are some studies that indicate an increase in symptoms after and surgery. These works report that dryness and foreign body sensation were the most common ones (Hovanesian et al. 2001; Solomon et al. 2004). In our study, we have found low frequency of symptoms after refractive surgery for both and. These previous studies used different questionnaires to assess the frequency and intensity of a large number of symptoms, and this may be one of the reasons for the differences between those works and ours in which the DEQ is used. It is known that 10% decrease in relative humidity produces 30% of tear evaporation and that this fact e9

triggers dry eye symptoms. Low relative humidity conditions (office environment, air-conditioned cars, airplane cabins and extreme hot or cold weather) negatively impact the tear film. On the contrary, an increase in humidity points to a more stable tear film, and, thus, more protection against dryness (McCulley et al. 2006). It is possible that environmental conditions, high relative humidity, in the geographic area where we conducted the study, could have caused a lower frequency of symptoms. It would be interesting to conduct further studies with refractive surgery patients on different environmental conditions to support this hypothesis. Refractive surgery causes nerve damage that decreases corneal sensitivity, and this hypoesthesia would decrease the symptoms. However, it has been reported that during the first 7 9 days after surgery, corneal sensitivity could increase, probably due to a local inflammatory reaction (Gallar et al. 2004).This decrease of sensitivity did not recover until 3 months to 1 year from surgery (Perez-Santonja et al. 1999; Benitez-del-Castillo et al. 2001). During this period of hypoesthesia, nerve endings that were damaged have been repaired and have grown back to innervate the entire corneal surface as originally. This growth is often abnormal, creating microaneuromas that transmit wrong signals or overreacted stimuli which may not exist or that are too mild to cause dryness, called dysesthesia (Gallar et al. 2004). Therefore, symptoms that relate to refractive surgery patients after 3 months, such as sensation of dry eye, do not necessarily reflect a real discomfort and may be due to dysesthesias provoked by a very slight dryness (Toda et al. 2006). There is only a recent study where tear film osmolarity has been measured after refractive surgery, finding an increase at 12 month post. Maybe a limitation of this study would be a period of the study. It would have been interesting to followup these patients for periods longer than 6 months, evaluating tear film osmolarity, in order to see whether or not they develop dry eye and assess potential dysesthesias. In this study, we did not evaluate tear osmolarity because the aim was not only to evaluate dry eye and dinucleotides levels relationship, but also its relationship with wound healing, where dinucleotides have a main role (Pintor et al. 2004). In conclusion, the levels of Ap 4 A are increased in refractive surgery patients during the first day after the surgery. This dinucleotide release may help in the wound healing process. During the first months, these patients do not show dry eye. 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spherical and astigmatic excimer laser photorefractive keratectomy. J Cataract Refract Surg 26: 1585 1589. Siganos DS, Popescu CN, Siganos CS & Pistola G (2002): Tear secretion following excimer laser in situ keratomileusis. J Refract Surg 18: 124 126. Solomon R, Donnenfeld ED & Perry HD (2004): The effects of on the ocular surface. Ocul Surf 2: 34 44. Toda I (2008): and the ocular surface. Cornea 27(Suppl 1): S70 S76. Toda I, Asano-Kato N, Komai-Hori Y & Tsubota K (2001): Dry eye after laser in situ keratomileusis. Am J Ophthalmol 132: 1 7. Toda I, Kato-Asano N, Hori-Komai Y & Tsubota K (2006): Dry eye after enhancement by flap lifting. J Refract Surg 22: 358 362. (2007): Methodologies to diagnose and monitor dry eye disease: report of the Diagnostic Methodology Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf 5: 108 152. Received on February 14th, 2013. Accepted on April 30th, 2013. Correspondence: Gonzalo Carracedo Rodríguez Department Optics II (optometry and Vision) Faculty of Optics C Arcos del Jalon 118 28032 Madrid, Spain Tel: 0034 913946859 Fax: 0034 913944677 Email: gonzalocarracedo@gmail.com e11