Investigations were performed according to the Declaration of Helsinki on Biomedical Research Involving Human Subjects. The epidemiological study was approved by Institutional Review Board of Kyoto Prefectural University of Medicine Review Board and after complete description of the study to the participants, written informed consent was obtained.

We carried out a cross sectional study of 1,025 Japanese (male: 542, female: 483) aged 17 to 73 years (average age 36.0 years, SD 10.0 years) who were engaging in VDT work. The VDT worker is characterized according to the occupational health management guideline for VDT workers [31]. The Japanese Society of Preventative Medicine administered dry eye examinations and questionnaires. Data were collected during the period November 2000 - February 2002. Tear film status was assessed by measuring the precorneal tear stability, lipid layer status and tear secretion. Precorneal tear film stability was assessed by measuring the fluorescein tear film break-up time (BUT). BUT is the interval between the last complete blink and the first appearance of a dry spot on precorneal surface tear film. Tear film lipid layer interferometry (DR-1; Kowa Co., Tokyo, Japan) was performed to evaluate the status of the tear lipid layer. DR-1 interferometry observes the specular reflected light from the tear surface. The classification of tear lipid layer patterns was evaluated by semiquantitative dry eye severity grading as previously reported [32]. (Materials and Methods S1) Examination of tear secretion was carried out by the standard Schirmer 1 test. The Schirmer test is a test to measure change in tear production by the observed wetting of a standardized paper strip placed over the inferior eyelid over a given period of time. All ophthalmic examinations were performed by ophthalmologists. Subjects were asked about the starting year/month of VDT use and duration of VDT use hour/day and day/month.

The amount of VDT used per year was calculated from starting year/month to consultation day of the study. Total hours of VDT use per month were calculated and the amount of time of VDT use per day was estimated. Average BUT and Schirmer score from both eyes were analyzed. Unconditional logistic regression analysis was used to calculate odds ratios as an estimate of the relative risk and 95 percent confidence intervals for the association between amount of VDT working duration and the risk of change in precorneal tear film stability, lipid layer status and tear secretion. The tear secretion were categorized into Schirmer score less than 5 mm and 5 mm or more, and precorneal tear film stability were categorized into tear film BUT less than 5 sec and 5 sec or more which is considered the definitive threshold value for dry eye [33], [34]. The tear lipid layer patterns were categorized into grade less than 3 and 3 or more. We examined the relationship between tear function and the amount of VDT use per year (<4, 4–8, 8–12, ≥12 years) and hours per day VDT (≤2, 2–4, 4–6 6–8, >8 hours). We studied linear trends by fitting categorical variables using median values for each category as continuous values in the model. The models were adjusted for age and gender.

We excluded participants who lacked data related to tear secretion (3), the amount of VDT use by year or time of VDT use per day (421). The data for the remaining 601 participants (335 males, 266 females) were used for the analyses of the relationship between amount of VDT use and tear function. Statistical analysis was performed using the JMP version 8 (SAS Institute Inc., USA).

Eight-week-old female Sprague-Dawley rats (Tokyo Laboratory Animal Science, Japan) were used for this study. At 1 week prior to the experiments, all animals were quarantined and acclimatized under the following general conditions: room temperature of 23±2°C, relative humidity of 60%±10%, an alternating 12-hour light–dark cycle (8 AM to 8 PM), and water and food available ad libitum. All animal experiments as follows were approved by the Animal Care and Use Committee of Ophtecs Corporation, and all procedures were performed in accordance with the Association of Research and Vision in Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.

The model and methodology used for the simulation of VDT has been previously published [14], [35]. Briefly, a series of treatments were performed under dry conditions, with room temperature of 23±2°C, relative humidity of 25%±5%, and a constant air flow of 2 to 4 m/s. After being placed on a swing, each rat was kept stationary for 7.5 hours per day between 9:00 AM and 5:00 PM. Lacrimal function and morphology were evaluated after undergoing 10 days of the swing procedure, unless otherwise mentioned.

In the study that examined shorter times on the swing, the amount of time the rat was on the swing was 4 and 6 hours, respectively. For the study that examined a modified resting pattern, various patterns were used during the 6-hour period the animals were on the swing. The variations used included 3 equal resting/swing periods, five 30-minute resting periods that were each followed by 1 hour on the swing, two 1-hour resting periods that were each followed by 2 hours on the swing, and one 2-hour resting period that was followed by 3 hours on the swing. Lacrimal functions were evaluated after 10 days for each of the different groups. In the study that examined stimulation of lacrimal function, tear fluid secretion was stimulated by subcutaneous injection of 0.75 mg/kg pilocarpine hydrochloride (Merck, Germany) every day after the allocated swing time. The modified Schirmer test was performed before pilocarpine injection on days 5 and 10. In the rehabilitation study, after the initial 10 days of the swing procedure, the animals were placed in cages and maintained under the same general conditions without any further time on the swing. In these animals, the recovery of the lacrimal function was evaluated on days 1, 5 and 10.

We used a modified Schirmer test [36] on the rats' eyes to measure tear fluid secretion under topical anesthesia induced with a 0.4% oxybuprocaine hydrochloride solution (Santen Pharmaceutical Co., Ltd., Japan). After 3 minutes of anesthesia, a phenol red thread (Zone-Quick; Menicon, Japan) was placed on the temporal side of the upper eyelid margin for 1 minute. The length of the moistened area from the edge was then measured to within 1 mm.

Changes in rat tear fluid secretion were measured under general anesthesia by intramuscular injection of an anesthesia cocktail containing ketamine and xylazine. Tear fluid secretion was stimulated by subcutaneous injection of 0.35 mg/kg to 1 mg/kg pilocarpine hydrochloride (Merck, Germany) after 15 minutes of general anesthesia. The Schirmer test was performed 5 minutes before and 30 minutes after pilocarpine injection.

Both exorbital LGs were excised and cut into fragments of 0.5 to 1 mm with a scalpel blade. The fragments were digested with 100 U/µg collagenase (CLS III, Worthington, USA) at 37°C for 30 minutes in 10 mL oxygenated saline solution (OSS, 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.81 mM MgCl₂, 1.01 mM NaH₂PO₄, 26.2 mM NaHCO₃, 5.6 mM dextrose [pH 7.4]), and then washed three times with OSS. After pre-incubation in OSS without carbachol for 30 minutes, the LG were incubated in OSS with 10⁻³ M carbachol (Cch) (Tokyo Kasei Kougyo, Japan) or without Cch for 90 minutes. During Cch stimulation, the culture medium was exchanged every 30 minutes and maintained at 37°C during the experiment. The protein concentration in the medium was measured using the Bradford reagent (Sigma-Aldrich, USA) with bovine serum albumin used as the standard. The protein secretion rate was calculated as a percentage of the before Cch stimulation value.

Animals were euthanized with an overdose of pentobarbital sodium, and their LGs were removed. For the hematoxylin and eosin staining, the LGs were fixed in 10% formalin. After dehydration, the LG specimens were embedded in paraffin, cross-sectioned, and stained. For the toluidine blue staining, the LGs were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), then postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer. After dehydration, the LGs were embedded in an epoxy resin, semithin sections were prepared and stained with toluidine blue. They were subsequently examined with a light microscope.

Deoxyribonucleic acid content of the LG was used to calculate cell density and total cell number. The exorbital LGs were excised after the rats were given a lethal dose of sodium pentobarbital. The LG tissue was homogenized in phosphate buffered saline containing 0.5% triton X-100. Propidium iodide (100 µg/ml, Invitrogen, USA) was added to the tissue suspension and fluorescein intensity was measured at an excitation of 530 nm and emission of 620 nm. Deoxyribonucleic acid from salmon sperm (Wako Pure Chemical, Japan) was used as the standard.

The removed LG was fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hour. Samples were then post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer at 4°C for an hour. The LG was dehydrated in graded ethyl alcohols and embedded in Epoc 812. An ultrathin section was cut using a RT-7000 (RMC, USA), stained with uranyl acetate and lead citrate, and then examined with transmission electron microscopy (JEM-100CX; JEOL, Japan).

For the rat studies we used the Student's t-test for comparison of the two groups and the Dunnett test for multiple comparisons. The Pearson product-moment correlation coefficient was used to evaluate association between LG morphology and tear secretion. Differences between measured variables were considered significant if the resultant P value was 0.05 or less.