Twelve female rhesus macaques (Mr Mulatta) aged 4–5 years were used in these experiments. Monkeys had free access to drinking water and were fed monkey food (12% calories from fat, 18% calories from protein, and 70% calories from carbohydrates; 200-300 g/day ). In addition, a daily ration of additional fruits, vegetables or supplements and various toys were also provided. All experimental protocols (AW2038) have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Primed Shines Bio-tech Co., Ltd. No animals were sacrificed for the purpose of this work. This study adhered to the principles of the Declaration of Helsinki and the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Association for Research of Vision and Ophthalmology.
Induction of dry eye
Monkeys were housed in an environmentally controlled room with relative humidity (RH) less than 15%, airflow of 12 L/min, and temperature of 21°C to 26°C, for 36 consecutive days. Dry eye was assessed clinically using corneal fluorescein staining on days 0, 14, 21, and 36 after dry eye induction.
Twenty-one days after dry eye induction, monkeys in the controlled environment room were randomly divided into two treatment groups (n = 6 monkeys/12 eyes in each group): (1) one group receiving topical normal saline as placebo, (2) topical Pred Forte 1% group (Allergan, Inc., USA). Two drops of normal saline and 1% Pred Forte were applied topically to both eyes of unanesthetized monkeys three times daily for 14 days (total, 42 doses). Dry eye was assessed clinically using fluorescein staining on treatment days 0, 7, and 14 (treatment day 0 equals post-induction day 21).
Corneal fluorescein staining (CFS)
Corneal fluorescein staining was performed on days 0 (baseline), 14, 21 and 36 after dry eye induction and on days 0, 7 and 14 of the treatment regimen. Ten microliters of 10% fluorescein liquid (Alcon Laboratories Inc., USA) was applied to the inferior-lateral conjunctival sac of the monkey, and after 10 minutes, corneal fluorescein staining was examined under the cobalt blue light using a slit lamp biomicroscope (TOKA TSL-5, Wenzhou Raymond Photoelectricity Tech. Co., Ltd., China). Punctate staining was assessed in a masked manner using the National Eye Institute scoring system, giving a score of 0-3 for each of five areas of the cornea17.
Tear Film Breakup Time (TFBUT)
Tear film break-up time was measured according to the guidelines set out in the International Dry Eye Workshop (DEWS) 2017 report11. Ten microliters of 2% fluorescein preservative-free solution were applied to the conjunctival sac with a micropipette. The monkeys were tricked into blinking their eyes three times by an ophthalmologist to ensure adequate mixing of the dye. The time interval between the last complete blink and the appearance of the first corneal black spot, indicating tear film breakdown, was measured using a stopwatch. The intensity of the background illumination was kept constant (cobalt blue light) and an integrated yellow filter was used to improve the visibility of the tear film over the entire cornea. The TFBUT was measured three times per eye and the mean value of the measurements was calculated.
The Schirmer I test after local anesthetic application was performed on days 0 (baseline) and 21 after dry eye induction to measure baseline tear secretion in monkeys, as previously described.4. Briefly, a 35 mm Schirmer test strip (Eickemeyer, Tuttlingen, Germany) was inserted into the inferior conjunctival fornix at the junction of the middle and lateral third of the lower lid margin. The eyelids were gently closed and the extent of wetting was measured after 1 and 5 min, respectively.
Collection of tears
Monkeys were laid on a table in the supine position after being anesthetized with an intramuscular injection of 10 mg/kg ketamine hydrochloride (Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd., China). Thirty microliters of phosphate-buffered saline solution was instilled into the lower fornix, and the monkeys were manually made to blink eight times. A total of 25 μL of tear lavage was collected with a micropipette from the lateral canthus. To minimize irritation to the ocular surface, we collected the tear wash immediately after application. The tear wash was placed in a 1.5 ml Eppendorf tube and stored at -80°C until further examination.
Measurement of IL-17, IL-2, TNF-α, IFN-γ
LEGENDplex™ bead-based immunoassays (BioLegend, USA) were used to measure IL-17, IL-2, TNF-α, IFN-γ levels in tears according to manufacturer’s instructions. Briefly, 25 μL of the solution consisting of the tear, standard, mixed beads, and buffer solutions were added to each tube. Then the tube was centrifuged at 800 rpm and incubated for 2 h at room temperature. After three washes to remove unbound proteins, 25 μL of corresponding detection antibodies were added to each tube. The tube was then centrifuged at 800 rpm and incubated for another hour at room temperature. After three washes to remove unbound detection antibodies, samples were read on a flow cytometer (Beckman CytoFLEX, USA) and then analyzed using LEGENDplex™ V8.0 software (BioLegend, USA).
Groups were compared by paired or unpaired two-sample t-tests. All values are expressed as the mean ± standard error of the mean (SEM). A p value