Animal Experiments

Adult male C57BL/6J mice (20–22 g) were purchased from Shanghai Laboratory Animal Center (SLAC, Shanghai, China). PPAR-α knockout (PPAR-α^{− / −}) mice with a C57BL/6J background were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and were bred at the Laboratory Animal Center of Xiamen University. Before using in the experiment, all mice adapted to the environment for a week, and were examined to ensure ocular surface health. SD was performed using a “stick over water” method in a standard laboratory setting, as previously described (Li et al., 2018). Two mice were housed per cage in standard laboratory conditions. In the middle of the cages, two 6-mm-diameter sticks were added, 4 cm above the bottom, and 6 cm apart. Water was added into the cage to 1 cm below the sticks. Animals were fed a standard diet and tap water ad libitum. When the mice were sleepy, they would lose balance and fall into the water, which awakened the animals. The SD time lasted for 20 h. Animals were observed once every 4 h to prevent death from drowning. After the SD period, each animal was gently dried using a hair dryer and blotting paper, and was transferred to its dry home cage. Sleep time was arranged in the noon, and any disturbance was forbidden. The room temperature was kept at 25 ± 1°C with a 12 h light:12 h dark cycle. Each animal was anesthetized using sodium pentobarbital [50 mg/kg, intraperitoneal (i.p.) route] before being humanely killed.

All animal experimental protocols were in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research guidelines and were approved by the Experimental Animal Ethics Committee of Xiamen University.

Each experimental animal was randomly assigned to one of seven treatment group conditions: 1. SD group (n = 57). Animals were sleep-deprived 20 h per day for 5 or 10 days and given water ad libitum. After SD, mice were transferred to their dry home cages, and disturbance was forbidden. 2. Control (Ctrl) group (n = 46). Animals were kept in standard home cages without SD or any other treatment. 3. Vehicle (Veh) group (n = 46). Animals were sleep-deprived for 20 h per day for 10 days. SD-induced mice were injected (i.p. route) with vehicle solvent (saline with 10% polyethylene glycol-400 and 10% Tween-80) twice a day (8 a.m. and 8 p.m.), from day 6 until day 10. 4. PEA treatment (PEA) group (n = 56). Animals were sleep-deprived for 20 h per day for 10 days. SD-induced mice were treated with PEA (3, 10, 30 mg/kg; i.p. route) twice a day (8 a.m. and 8 p.m.), from day 6 until day 10. 5. MK886 treatment (MK886) group (n = 10). SD-induced mice were treated with PPAR-α antagonist MK886 (15 mg/kg; i.p. route) twice a day (8 a.m. and 8 p.m.), from day 6 until day 10. 6. PEA treatment with MK886 (PEA + MK886) group (n = 10). SD-induced mice were injected with PPAR-α antagonist MK886 (15 mg/kg; i.p. route) twice a day (8 a.m. and 8 p.m.), from day 6 until day 10. Twenty minutes later, the mice were treated with PEA (30 mg/kg, i.p.). 7. PPAR-α knockout mice (PPAR-α^−/−) group (n = 11). Animals were kept in standard home cages without SD, and the other treatment was consistent with the description above.

A total of 20–30 mg frozen LG tissue samples were homogenized in 2 ml mixtures of methanol/water (1:1, vol:vol) containing 1 nM heptadecenoylethanolamide (17:1 FAE) as an internal standard. Endogenous FAEs were extracted with 4 ml chloroform, vortexed for 1 min, and then centrifuged for 10 min at 4°C and 3000 × g. Most of the FAEs were separated during the chloroform phase. The organic phase was collected and evaporated to dryness under an N₂ stream using a nitrogen evaporator (Beijing, TongtaiLian Technology Co., Ltd.). The dried lipids were resuspended in 1 ml chloroform. Solid-phase extraction was performed by loading the lipid solution into its respective silica gel column. The samples were eluted with 3 ml chloroform/methanol (9:1, vol:vol). The purified FAEs were dried under an N₂ stream, and then redissolved in 100 μl methanol for HPLC/mass spectrometry (MS²) analyses.

The samples were analyzed using an 1100-HPLC system (Agilent, Shanghai, China) equipped with an Applied Biosystems 3200 triple-quadrupole linear ion trap mass spectrometer (Applied Biosystems, Concord, Canada). The elution gradient for the mobile phase was: 90% methanol for the first 5 min, followed by a linear gradient of 90% to 100% methanol for 5 min, and then maintenance at 100% methanol for 20 min. The HPLC flow rate was 0.7 ml/min. The column temperature was 40°C. Multiple reaction monitoring transitions in positive ion atmospheric pressure chemical ionization mode were performed for ion detection: AEA, m/z 348.00/62.00; OEA, m/z 326.10/62.00; PEA, m/z 300.2/62.00; and 17:1 FAE, m/z 313.1/62.00.

Phenol red cotton thread (Zone-Quick; Yokota, Tokyo, Japan) was used to absorb aqueous tear production for measurement. Each test was conducted in the standard environment at the same time by the same operator. Briefly, the mouse lower eyelid was pulled down gently to expose the conjunctival sac. The thread was then placed on the lower conjunctival fornix near the lateral canthus at about one-third of the length of the lower lid. After 15 s, the thread was removed, and the red wetted length was measured in millimeters. After the test, both eyes were closed to avoid excessive exposure leading to ocular surface irritation.

Eyes with ocular adnexa were surgically excised and fixed in 4% paraformaldehyde for 24 h. After dehydration in gradient alcohol, the tissues were embedded in paraffin and cut into 8 µm sections. Dimethylbenzene was used for deparaffinization. After soaking in double-distilled water for 10 min, the tissue sections were stained with periodic acid for 10 min. Schiff reagent (Leica Biosystems, Nussloch, Baden, Germany) was used to stain the sections for 8 min after rinsing with double-distilled water for 10 min. Subsequently, the sections were soaked in double-distilled water for another 3 min and were stained with hematoxylin for 35 s. During counting, the goblet cells in the superior and inferior conjunctivae were viewed using a light microscope (Eclipse 50i, Nikon, Tokyo, Japan).

The LG samples were placed in 4% paraformaldehyde at 4°C for fixation. After dehydration and clearing, the tissues were embedded into paraffin wax and cut into 6 µm sections. The tissue sections were deparaffinized, rehydrated, and stained for morphological examination using hematoxylin and eosin. In order to ensure the reliability of the data, we kept the placement position consistently when the LG is embedded. The slice is guaranteed to have the same cross-sectional direction when sectioning. In addition, three slides were taken from the front, middle, and last parts of each sample during sectioning. The morphological differences of LGs were compared by the ratio of the mean area of individual acini in each group to the control group of acinar area under the same magnification micrographs.

LG tissues were collected from the mice and immediately flash-frozen in optical coherence tomography (OCT, SAKURA Tissue-Tek, Torrance, CA, USA) with liquid N₂. The tissues were cut into 6-µm-thick sections using a cryostat microtome (CM1850 UV, Leica Microsystems, Wetzlar, Germany). The sections were air-dried for 10 min at room temperature, fixed with 10% buffered formalin for 20 min, and then washed with 1× PBS solution three times (5 min each time). Approximately 1 ml oil red O (ORO) working solution was added to completely cover the sections, and they were incubated at room temperature for approximately 10 min. The sections were submerged in hematoxylin for 45 s, and thereafter rinsed under running water for 10 min. The ORO stain results were examined using light microscopy (Eclipse 50i, Nikon, Tokyo, Japan).

The LG tissues were immediately collected after the mice were euthanized; the tissues were fixed in a 2.5% solution of glutaraldehyde buffer to preserve the fine structure. The tissues were cut into small pieces (1 × 1 × 2 mm) and fixed overnight at 4°C. They were then washed with three times (15 min each time) with cold PBS and secondarily fixed in 1% osmic acid for 2 h at 4°C. After dehydration with gradient ethanol and infiltration with Spurr’s resin, the fixed tissues were encased in hardened blocks to be thin-sectioned using an ultramicrotome (Leica EM UC6 and Reichert Ultracut S, Leica Microsystems GmbH, Wetzlar, Germany). The thin LG sections were counterstained and then examined and photographed using a transmission electron microscope (TEM, JEM2100HC, JEOL, Tokyo, Japan).

Corneal epithelial permeability was assessed using Oregon Green Dextran (OGD, 70 kDa, Invitrogen, Eugene, Oregon, USA) for both eyes in the mice from each group. Briefly, 0.5 μl OGD (50 mg/ml) was applied to the mouse cornea for 1 min. The mice were then euthanized, and the eyes were rinsed with 1 ml saline (five times). Digital pictures were photographed using a multizoom fluorescence microscope (470 nm excitation and 488 nm emission wavelengths, AZ100, Nikon, Tokyo, Japan). The mean fluorescence intensity of corneal OGD staining in the digital images was calculated for a 3-mm-diameter circle in a fixed central cornea region using analysis software (NIS Elements, version 4.1, Nikon, Melville, NY, USA).

Corneal sensitivity was quantified by using a Cochet-Bonnet esthesiometer (Luneau, Paris, France). The pressure applied to the center of the cornea was proportionate to the inverse of the esthesiometer nylon filament length. That is, the longer the nylon filament, the lower the pressure exerted on the apex of the cornea. When the force exerted perpendicularly to the center of the cornea was perceived by the mouse, a blink response was evoked. Determination of the threshold to stimulation was made using a 0.12-mm-diameter filament with a maximum length of 60 mm. Five consecutive stimulus presentations were conducted for each eye. When three or more blink responses were observed in the test, a positive result was considered, and the length was recorded. If the threshold could not be detected, the nylon filament length was reduced in 5 mm increments to increase stimulus intensity.

The total RNA was extracted from LG tissues using TriPure isolation reagent (Roche, Shanghai, China) according to the manufacturer’s protocol. Total RNA was inverted into cDNA using a ReverTra Ace qPCR RT kit (TOYOBO, Shanghai, China). The amount of cDNA required for running PCR was added according to the Fast Start Essential DNA Green Master (Roche, Shanghai, China) instructions. Quantification of mRNA expression was performed using SYBR Premix Ex TaqTMII (Takara, Dalian, China) and a LightCycler 96 System (Roche, Shanghai, China). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard to normalize the relative expression of target genes.

The primers were designed and synthesized based on gene sequences available in the GenBank database. The mouse gene sequences were: NAPE-PLD, forward primer (F): 5’-TGGCTGGGACACGCG-3’, reverse primer (R): 5’-GGGATCCGTGAGGAGGATG-3’; fatty acid amide hydrolase (FAAH), (F): 5’-GCCTCAAGGAATGCTTCAGC-3’, (R): 5’-TGCCCTCATTCAGGCTCAAG-3’; NAAA, (F): 5’-GACTCCGCCTCTCTTCAACG-3’, (R): 5’-ACCATCCCGAGTACCCACTG-3’; PPAR-α, (F): 5’-AGAGCCCCATCTGTCCTCTC-3’, (R): 5’-ACTGGTAGTCTGCAAAACCAAA-3’; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (F): 5’-ACCACGAGAAATATGACAACTCCC-3’, (R): 5’-CCAAAGTTGTCATGGATGACC-3’; stearoyl-CoA desaturase-1 (SCD1), (F): 5’-ATCGCCCCTACGACAAGAAC -3’, (R): 5’-AACTCAGAAGCCCAAAGCTCA-3’; carnitine palmitoyl-transferase 1α (CPT1α), (F): 5’-GACTCCGCTCGCTCATTCC-3’, (R): 5’-CACCAGTGATGATGCCATTCTTG-3’; lipoprotein lipase (LPL), (F): 5’-AGGGCTCTGCCTGAGTTGTA-3’, (R): 5’-AGAAATCTCGAAGGCCTGGT-3’; acyl-CoA oxidase 1 (ACOX1), (F): 5’-TCGAAGCCAGCGTTACGAG -3’, (R): 5’-GGTCTGCGATGCCAAATTCC-3’; acetyl coenzyme A carboxylase 1 (ACC1), (F): 5’-TGCAGGTATCCCCACTCTTC-3’, (R): 5’-TTCTGATTCCCTTCCCTCCT-3’; acetyl coenzyme A carboxylase 2 (ACC2), (F): 5’-TTTCTGATGTGCTGGAATGG-3’, (R): 5’-GACTGTGTGTGCTCGTGGTT-3’.