Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Summary

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Abstract

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Introduction

Dry eye has become the most common ocular surface disease worldwide, with an estimated 5% to 50% of the population suffering from the disease¹. Eye dryness and discomfort caused by dry eye, if persistent, can lead to visual disturbances and unstable tear films, further leading to ocular surface damage². Some patients with dry eye may even suffer from chronic pain².

According to the 2017 TFOS DEWS II Dry Eye Consensus, dry eye is a complex condition that involves multiple factors and is characterized by the disruption of the natural balance of the tear film. This condition is accompanied by a range of ocular symptoms, instability of the tear film, increased osmolarity of the tears, inflammation and damage to the ocular surface, and neuropathic pain, which are crucial in understanding the disease's progression³. In this feedback loop, dry eye can cause damage to corneal epithelial cells, decrease corneal stromal thickness and endothelial cell density, trigger immune cell activation and aggregation on the corneal surface, reduce mucin secretion by goblet cells, and cause immune dysfunction in the lacrimal gland, further exacerbating the condition^{4,5,6,7,8,9,10}. The consensus delineates three primary forms of dry eye syndrome: aqueous deficiency dry eye, evaporative dry eye, and a combination of both, known as mixed dry eye³. Aqueous deficiency dry eye is associated with structural and functional changes in the lacrimal glands¹¹. These alterations include acinar atrophy, ductal occlusion that blocks the flow of tears, lymphocyte infiltration indicating an immune response, and a reduction in the secretion of proteins that are essential for maintaining the health and stability of the tear film^12,13.

The lacrimal gland is a tubular exocrine gland that is responsible for the production of water components in the tear film^13,14, including water, electrolytes, and proteins, and contributes to the maintenance of the stability of the ocular surface microenvironment^15,16. The health and visual clarity of the eye depend on the presence of the tear film, which forms a critical optical layer on the surface of the cornea, ensuring smooth lubrication of the cornea and conjunctiva¹⁷. The tear film promotes the metabolic activity of ocular surface cells and acts as a cleaner, effectively removing impurities and potential irritants from the eye surface, thereby maintaining the physiological balance and comfort of the eye^18,19.

In rodents, such as rats and mice, the lacrimal glands consist of two lobes: the internal and external orbital lobes. The external orbital lobe is located below the ear, and its functional part is connected to the eye by an extended duct. This duct engages with the lacrimal gland lobe in the orbit before reaching the eye and is involved in tear production²⁰. The internal orbital lobe, also known as the intraorbital lacrimal gland, is located under the bulbar conjunctiva of the outer canthus. The ILG is smaller than the ELG. Impaired lacrimal gland function can lead to aqueous deficiency dry eye, which, if left untreated, can progress to corneal ulcers and vision loss²¹.

Animal models of aqueous deficiency dry eye can be categorized into three intervention methods: surgical induction models, drug-induced models, and transgenic models. The surgical induction model involves the removal of the ELG. However, this model is not stable due to the presence of ILG²². The systemic injection of the cholinergic receptor blocker scopolamine is common in the drug-induced model²³. Topical applications of atropine²⁴ and benzalkonium chloride²⁵ can also reduce mucin and tear secretion. Concanavalin-A can be injected locally into rabbit lacrimal glands to cause immune lacrimal adenitis, thereby establishing a model of aqueous deficiency dry eye²⁶. Additionally, transgenic animals, such as NOD, Aly/aly, NFS/sld, IQI/Jic, Id3 KO, and other mouse strains, can mimic the symptoms and phenotypes of primary Sjögren syndrome²⁷ to replicate aqueous deficiency dry eye.

In this study, the core experimental design involved a surgical procedure to painlessly and precisely remove the ELG and the ILG of the mouse. These lacrimal glands are essential for maintaining eye moisture and lubrication; their removal results in a significant reduction in tear secretion, mimicking aqueous deficiency dry eye in mice. This approach allows for the observation and analysis of mouse behavior, physiological responses, and ocular tissue changes in the dry eye state, providing a key experimental basis for pathology studies and the evaluation of treatment options for dry eye disease.

Protocol

Female C57BL/6 (C57) mice, aged 7-8 weeks, were used in this study. All procedures adhered to the ARVO guidelines for the use of animals in ophthalmic and vision research and were approved by the Animal Ethics Committee of Guizhou Medical University (Approval No. 2305193). No ocular surface lesions were observed under a slit lamp examination.

1. Preoperative steps

1. Sterilize the instruments using a rapid sterilizer: needle holders, a 5-0 suture needle with thread, ophthalmic surgical scissors, and forceps.
2. Anesthetize the mouse with 1.25% Avertin via intraperitoneal injection at a dose of 0.3 mL/100 g.
3. After anesthesia is complete, place the mouse in the lateral decubitus position and remove the hair from the surgical area.
4. Disinfect the skin and hair around the eyes with iodophor.
5. Establishment of the dry eye model
   1. Place the mouse in the lateral decubitus position. Expose the surgical area under an operating microscope.
   2. Clamp the skin on the right side of the mouse's face with toothed forceps and make an incision at the midpoint of the line intersecting the external auricle and the mandibular line to the inner corner of the eye. Expose the muscle tissue; remove the ELG located on the muscle carefully.
   3. Extend the skin incision to the inner corner of the eye, bluntly separate the muscle, and locate the light red gland beneath the muscle. Peel off and remove the ILG.
   4. Suture the incision using 5-0 sutures with the help of a needle holder, ophthalmic surgical scissors, and forceps.
   5. Apply ofloxacin ointment at the end of the operation to prevent infection.
6. After removing the unilateral ELG and ILG, place the mice in an environment with a rhythmic 12:12 h light-dark cycle with free access to food and water. This creates a unilateral dry eye model, while the contralateral eye with internal and external lacrimal glands serves as the normal control group.

2. Measurement of tear production

1. Anesthetize the mouse with 1.25% Avertin intraperitoneally at a dose of 0.3 mL/100 g.
2. Take a well-packaged phenol red thread for tear measurement.
3. Gently pull down the lower eyelid to be measured. Position the top of the phenol red thread into the inner and outer thirds of the lower eyelid, initiating the timer promptly.
4. Perform the test on one eye at a time for a duration of 15 s.
5. After the allotted time, carefully retract the lower eyelid and remove the phenol red thread downward with caution.
6. Utilize the scale provided on the outer bag of the phenol red thread to measure from the top of the thread to the entirety of the red portion.
7. Employ an electronic stopwatch to accurately record the duration of the test.

3. Preparation of freezing tissue slices

1. Perform rapid cervical dislocation on the mice following asphyxiation with CO₂.
2. Utilize scissors and forceps to carefully extract the eyeballs.
3. Embed the eyeballs in optimal cutting temperature compound and utilize a frozen microtome to obtain freezing tissue slices of 5-7 µm thickness²⁸.
4. Store all slides in a specimen box at room temperature for approximately 30 min. Subsequently, transfer them to an ultra-low temperature freezer at -80 °C.

4. Hematoxylin and eosin (H&E) staining

1. Remove frozen tissue slices from the ultra-low temperature freezer, lay them flat in a tissue box, and dry them in a fume hood.
2. Wash slices with 1x PBS, cover for 5 min, and then remove the PBS. Cover the specimen with 4% paraformaldehyde, ensuring the lid is closed to prevent evaporation. After 15 min at room temperature, rinse the specimens for 3 x 5 min in 1x PBS.
3. Place the specimen in a rack and immerse it in 0.5%hematoxylin dye for 5 min.
4. Rinse the specimen in tap water for 5 min to wash off any excess dye. Subsequently, immerse it in 0.1% hydrochloric acid alcohol for 1-2 s to facilitate differentiation.
5. Place the specimen holder in tap water for 15 min to remove the non-specific staining.
6. Drain excess water and immerse the specimen in 0.05% eosin staining solution for 3 min.
7. Wash the specimen in tap water and immerse it sequentially in 75%, 80%, 95%, and two rounds of 100% alcohol for 2 min each.
8. Immerse the specimen holder in two rounds of xylene for 2 min each.
9. Place the holder in a fume hood to dry and mount the specimen with mounting medium, avoiding damage and bubbles.
10. Store the stained specimens in a box at room temperature and photograph them using a biomicrography system.

5. Immunofluorescence staining

1. Retrieve the specimen from the freezer, lay it flat in the box, and dry it in the hood.
2. Wash the specimen with 1x PBS, cover for 5 min, and aspirate the excess liquid. Cover the specimen with 4% paraformaldehyde, add water to the immunohistochemical wet box, and close the lid to prevent evaporation. Allow it to sit at room temperature for 15 min; then, rinse the specimen 3 x 5 min in 1x PBS.
   NOTE: Handle the specimen with care during this process.
3. Remove excess PBS from the specimen surface and gently wipe the residual liquid around the tissue section with filter paper. Cover the specimen surface with cell permeabilization solution (0.2% Triton X-100) and incubate at room temperature for 20 min to permeabilize the membrane.
4. Remove the permeabilization solution and rinse the specimen 3 x 5 min with 1x PBS.
5. Pour out the PBS from the specimen surface and gently wipe residual liquid around the tissue section with filter paper. Cover the specimen with prewarmed blocking solution (2% BSA). Close the lid to prevent evaporation and incubate at room temperature for 1 h.
6. During the blocking process, select the appropriate antibody according to the experimental needs, prepare the primary antibody with immunofluorescent antibody diluent (1% BSA) according to the recommended ratio, and centrifuge the prepared primary antibody in a 4 °C centrifuge at 2,000 × g, 10 min.
   NOTE: All of the above are operated on ice or at low temperatures. It is recommended to use about 120 µL of primary antibody per sample.
7. Discard or pour off the primary antibody. Wash the specimen 3 x 10 min with 1x PBS buffer. After pouring off the PBS, rinse the specimen with fresh 1x PBS. Ensure that the PBS covers the slide during the first rinse.
8. While the primary antibody is being rinsed, select the suitable secondary antibody based on its category. Dilute it with 1% BSA diluent at a 1:300 ratio. Spin the prepared antibody in a centrifuge at 4 °C, 2 000 × g for 10 min. Perform the entire process on ice or at a low temperature in the dark.
9. Rinse the slide with PBS, gently wipe the liquid around the specimen with filter paper, use a suction pump to absorb any remaining liquid, and quickly add the prepared secondary antibody to the specimen. Cover it and incubate at room temperature for 1 h. Ensure all processes are conducted in the dark.
10. Discard the secondary antibody, rinse with 1x PBS buffer, and wash 3 x 10 min. Ensure the PBS completely covers the slide. Handle the specimen gently to avoid rinsing it out.
11. Pour off the 1x PBS, wipe away excess liquid with filter paper, use a suction pump to remove any remaining liquid, and mount the specimen with a mounting medium containing DAPI. Place the cover on the slide plate and store it in a 4° C freezer, protected from light with tin foil. Take care to avoid damaging the specimen and creating air bubbles.
12. Photograph the stained sample using a laser confocal or upright fluorescence microscope system. Ensure that the imaging is done within a week to prevent fluorescence quenching.

6. Extraction of corneal mRNA

1. Perform rapid cervical dislocation on the mice following asphyxiation with CO₂. Then, utilize scissors and forceps to carefully extract the eyeballs, wash them 3x in 1x PBS, and place the eyeball in a Petri dish containing 1x PBS.
2. Under the microscope, utilize scissors and forceps to cut the eyeball from the posterior pole of the eyeball to separate the lens. Clean up the excess sclera and iris, but leave the 0.5 mm white sclera and the entire cornea. Extract the mRNA from the mouse eyeballs using an RNA extraction kit according to the instructions.

7. Reverse transcription PCR

1. Before performing reverse transcription, determine the concentration of RNA with a UV spectrophotometer.
2. Take 1 mg of RNA based on the measured RNA concentration. Perform the reverse transcription reaction using a reverse transcription kit.
3. After adding the reaction components, add the mixture to a 200 μL centrifuge tube, mix with gentle shaking, centrifuge at low speed to avoid air bubbles, and immediately put it into a normal thermal cycler for reaction.
4. Analyze the cDNA from the reverse transcription process directly by real-time PCR or store it in a -20 °C freezer.

8. Real-time quantitative PCR reactions (qRT-PCR)

1. Set up a 10 µL reaction system according to the instructions provided with real-time PCR kit²⁹: 3.6 μL of DEPC-treated water, sense primer 0.2 μM, anti-sense Primer 0.2 μM, 1 μL of cDNA, 5 μL of Mix. See Table 1 for details about the primers.
2. Depending on the grouping of experiments, add the reaction system to a 96-well plate or eight-strip tube dedicated to qRT-PCR. Once the qRT-PCR system is configured, make sure the mixture is well mixed and use a tabletop centrifuge for brief centrifugation to shake off the liquid from the tube wall. Always check the tube for air bubbles before qRT-PCR.
3. Perform the reaction in triplicate and calculate the average Ct (cycle threshold).

Results

To investigate the effects and underlying mechanisms of aqueous deficiency dry eye on the ocular surface, we established a mouse model of aqueous deficiency dry eye by surgically removing both the ELG and ILG (Figure 1A). Following a 2-week period post tear gland resection, tear secretion in the dry eye (after removal of lacrimal glands) mice notably decreased compared to the normal group (Figure 1B,C). Evaluation of the cornea via slit lamp mic...

Discussion

We successfully established a mouse model of aqueous deficiency dry eye disease by surgically excising both the ELG and ILG, aiming to investigate changes in the ocular surface under conditions of aqueous deficiency dry eye disease. We meticulously documented the morphological and physiological alterations observed in this mouse model of dry eye resulting from the double excision of the ELG and ILG. Two weeks post modeling, we observed and sampled the established mouse model of dry eye.

Consi...

Materials

Name	Company	Catalog Number	Comments
10 mL syringe	Zhejiang KDL Medical Devices Co., Ltd. China
5-0 Suture Needle	Suzhou 66 Vision Co., Ltd. China
Absolute ethanol	Shanghai Sinopharm Chemical Reagent Co., Ltd., China
Alexa Fluor 488, Donkey anti mouse IgG	Invitrogen	811493
Alexa Fluor 594, Donkey anti rabbit IgG	Invitrogen	A21207
Anti-Keratin 12 antibody EPR17882	Abcam	ab185627
Anti-PAX6 antibody	Abcam	ab5790
Autoclave	Hirayama. Japan
C57BL/6 mouse	Shanghai Slack Laboratory Animal Co., Ltd.
Centrifuge at room temperature	Eppendorf. Germany
ddH2O	Shanghai Bioengineering Co., Ltd. China
Electronic balances	Shanghai Auhaus Biotech Co., Ltd. China
Fluorescence inverted phase contrast micrography system	TE-2000U,Nikon. Japan
Freeze the cassette	Jiangsu Shitai Experimental Equipment Co., Ltd. China
Freeze the slicing blade	Xiamen Taijing Biotechnology Co., Ltd. China
H-1200 with DAPI mounting medium	Xiamen Juin Biotechnology Co., Ltd. China		mounting medium containing DAPI
H-5000 Tablet Mountant	Vector. USA		mounting medium
HCl	Shanghai Sinopharm Chemical Reagent Co., Ltd., China
Hematoxylin-eosin stain kit	Auragene. USA
Iodine
Ki-67 antibody	Abcam	ab16667
liquid nitrogen	Xiamen Yidong Gas Co., Ltd. China
Microscopic needle holder	Suzhou 66 Vision Co., Ltd. China
Microscopic toothed forceps	Suzhou 66 Vision Co., Ltd. China
Microscopic toothless forceps	Suzhou 66 Vision Co., Ltd. China
Model 3050 frozen slicer	Leica, Deerfield, IL. USA
OCT	Shanghai Maokang Biotechnology Co., Ltd. China
Ofloxacin ointment
Ophthalmic sodium fluorescein test strips	Tianjin Jingming New Technology Development Co., Ltd. China
Paraformaldehyde powder	Sigma. USA
PBS
Phenol red cotton thread
Rapid sterilizer sterilizer	Suzhou 66 Vision Co., Ltd. China
Recombinant Human SPRR1b protein	Abcam	ab167925
Refrigerated tabletop centrifuge	Eppendorf. Germany
Slide holders
Slit lamp	Topcon. Japan
Specimen box	Lambolide (Fuzhou) Biotechnology Co., Ltd. China
Tert-Amyl alcohol	Shanghai. Macklin Biochemical, China	A800283	500 mL
Tribromoethanol
Triton X-100	Sigma. USA
Ultra-low temperature freezer	Thermo Fisher Scientific. USA
Upright fluorescence micrograph	Leica DM2500. USA
Upright normal biomicrography system	Eclipse 50i, Nikon. Japan
Xylene	Shanghai Sinopharm Chemical Reagent Co., Ltd., China
Zeiss Surgical Microscope	VISU150, Carl ZEISS. Germany