Subjects
This research was implemented in accordance with the requirements of the Declaration of Helsinki and the protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Army Medical University. The clinical trial registration number is ChiCTR2000038673.
All subjects were 18-30 years old and agreed to participate in this study and signed an informed consent. The diagnostic criteria for dry eye was the average non-invasive tear breakup time (A-NITBUT) < 10s according to the Tear Film and Ocular Surface Society Dry Eye Workshop Ⅱ(DEWS Ⅱ) [10]. Subjects were excluded if they had allergic conjunctivitis, Sjögren's syndrome, lacrimal obstruction or other ocular surface diseases. Subjects were also excluded if they had dry eye treatment or used contact lenses within one month, if they had a history of eye trauma or surgery within one year or subjects with serious systemic diseases who were not suitable for strenuous exercise.
Study design
The test was divided into two parts in order to avoid the possible influences of Schirmer test on the subsequent measurements of signs associated with dry eye. In total, 34 eyes of 17 dry eye patients and 34 eyes of 17 normal subjects were observed before and at 0, 30 minutes after AE in part 1. The evaluated variables included Schirmer Ⅰ test and six tear compositions. A total of 30 eyes of 15 dry eye patients and 30 eyes of 15 normal subjects were observed in part 2. The evaluated variables included visual acuity before and at 0, 30, 60 minutes after AE, signs associated with dry eye before and at 10, 20, 40 minutes after AE including tear meniscus height (TMH), first non-invasive tear breakup time (F-NITBUT), A-NITBUT, tear film lipid layer thickness (LLT), incomplete and complete blinks and partial blink rate (PBR). All the subjects were required to fast throughout the test period because eating have an effect on tear secretion [11].
AE protocol
The AE protocol was defined as jogging for 30 minutes. The venue was outdoors, the ambient temperature was 25-27°C while the humidity was 50-60% during the test. According to the 6-20 Rating of Perceived Exertion Scale, the target heart rate was set to 64-76% of the maximum heart rate in order to achieve moderate exercise intensity [12]. The maximum heart rate was defined as 220 minus age [12]. The measuring time was between 18:00 and 19:00.
Test items
OSDI
The Ocular Surface Disease Index (OSDI) questionnaire was used to quantify the subjective symptoms of dry eye [13]. The symptoms and environmental triggers for dry eye in the past week were assessed. OSDI score ≥13 was considered positive. The score range was 0-100. The higher the score, the more severe the symptoms.
Schirmer Ⅰ test
The researcher wore gloves and placed the Schirmer test strip (35mm; DSA Exports, India, without fluorescent agent) at the outer 1/3 of the lateral eyelid margin without using anesthetics. Both eyes were tested simultaneously and the eyes remained closed during the procedure. The strip was removed after 5 minutes and the length of wetted part up to the indentation line was recorded. Each test was carried out in a quiet and dark environment.
Analysis of tear compositions
Schirmer test strips were used to collect the tears. The test strips were stored in a refrigerator at -80°C until testing. Amount of PBS buffer to be added to each strip was calculated by multiplying the wetted length of the Schirmer test strip (Schirmer test reading + about 5mm of the head of strip without scale) by 100μl. After homogenizing, the samples were centrifuged at 2-4℃ for 15 minutes (2000 rpm). In total, 10μl of the supernatant was collected for analysis after precipitation. Six compositions of the tear were determined using ELISA kit (Jiangsu Meimian, China). The compositions tested included dry eye diagnostic factor lactoferrin [14] and matrix metalloproteinase-9 (MMP-9) [15], dry eye inflammation marker IL-6 [16], oxidative stress marker 8-hydroxy-2’-deoxyguanosine (8OHdG) [17], (O-acyl)-ω-hydroxy fatty acids (OAHFA) which is closely related to tear film stability [18], and Mucin 5 subtype AC (MUC5AC) which is considered to be the most abundant secretory mucin in human tears [19]. The absorbance was measured at 450 nm with a multifunctional microplate reader (Labsystems Multiskan MS, Finland).
Measurements of signs associated with dry eye
Keratograph 5M (OCULUS, Wetzlar, Germany) was used to measure TMH, F-NITBUT and A-NITBUT. Each measurement was measured twice by the same ophthalmologist and average measurements was recorded. Images with unclear boundaries of the tear meniscus were deleted to avoid affecting image analysis. LipiView LVI-1001 (TearScience, Inc., Morrisville, North Carolina) was used to measure LLT and shot 20s video which automatically recorded incomplete and total blinking [20]. Number of complete blinks (number of total blinks minus incomplete blinks) and PBR (number of incomplete blinks / total blinks) were obtained using simple calculation.
Visual acuity
ETDRS chart was used to measure the best corrected visual acuity.
Statistical analysis
Statistical analyses were performed with SPSS version 20.0 software package (IBM Corp., Armonk, NY, USA). Data was expressed as median (25% interquartile, 75% interquartile) or mean ± standard deviation. The Mann-Whitney U test was applied to compare the age and OSDI score between two groups. The Schirmer Ⅰ test, tear compositions, signs associated with dry eye and visual acuity at different time points were compared using two-way repeated measures analysis of variance. Bonferroni correction was used for multiple comparisons. The level of significance was set at P < 0.05.
Sample size
The sample size was calculated using PASS software (version 15.0, NCSS, LLC). According to the results of the A-NITBUT in preliminary experiment, the sample size was estimated by adopting the significance level (α) as 0.05, the desired power (1-β) as 0.85, the autocorrelation coefficient as 0.7 and the standard deviation of the population of 5.1. The estimated sample size was at least 29 eyes in each group.