A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

Podsumowanie

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

Streszczenie

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal tear production can lead to a disturbance in the ocular surface environment, including corneal damage and inflammation. In severe cases, ADDE can cause vision loss or even blindness. Currently, dry eye treatment is limited to eye drops or physical therapy, which can only alleviate eye discomfort symptoms and cannot fundamentally cure dry eye syndrome. To restore the function of the lacrimal gland in dry eye, we have created an animal model of lacrimal gland dysfunction in rats induced by scopolamine. Through the comprehensive evaluation of the lacrimal gland, corneas, conjunctivas, and other factors, we aim to provide a full understanding of the pathological changes of ADDE. Compared with the current dry eye mouse model, this ADDE animal model includes a functional evaluation of the lacrimal gland, providing a better platform for studying lacrimal gland dysfunction in ADDE.

Wprowadzenie

By 2021, approximately 12% of people are significantly affected by dry eyes¹, making it one of the most common chronic eye diseases. Dry eye can be divided into two types: aqueous-deficient dry eye (ADDE) and evaporative dry eye (EDE)², depending on the different factors that affect the disease. ADDE is further divided into Sjögren's syndrome (SS) and non-SS, but the majority of dry eye patients are non-SS patients in clinical³. Chronic dry eye symptoms seriously affect the visual quality of patients. Currently, the conventional treatment of DED involves the application of artificial tears to lubricate the ocular surface and physical therapy of the eyelids. However, dry eye syndrome may not offer a complete cure in many cases. Therefore, studying the pathogenesis of dry eye disease is crucial for the development of new therapies and drugs. Animal models of dry eye syndrome provide a foundation for further research.

There are many ways to construct animal models of dry eye syndrome⁴, including changing tear secretion levels by altering hormone levels. For example, removing the testes of rats can reduce androgen secretion, increase tear secretion, and decrease the concentration of free secretory component (SC) and IgA in tears^5,6. Another method is to indicate autoimmune reactions in the lacrimal gland by removing the eye surface nerves that control the gland. Additionally, directly reducing tear secretion can be achieved by surgically removing the lacrimal gland⁷. Changing environmental conditions can also accelerate tear evaporation. For example, culturing animals in low humidity and dry ventilation conditions can establish a model of excessive evaporative dry eye⁸, which can be combined with other methods to increase the severity of dry eye. The main drugs used to induce dry eye experimental models are atropine and scopolamine⁹. As parasympathetic inhibitors, both can induce pharmacological blockade of cholinergic (muscarinic) receptors in the lacrimal gland and inhibit tear secretion. Compared with dry eyes caused by atropine muscle injection¹⁰, scopolamine has a stronger inhibitory effect on secretion glands, a longer duration of drug action, and weaker effects on cardiac, small intestinal, and bronchial smooth muscles. It is one of the most mature drugs for dry-eye animal models.

Different methods can be used to induce dry eye with scopolamine, such as subcutaneous injection, drug pump, or patch application^4,11,12. In order to reduce the frequency of drug administration to experimental animals, many researchers apply transdermal patches to the tails of mice or use drug pumps. However, both of these methods have limitations. For example, the absorption of transdermal patches needs to take into account the individual absorption of mice, which can lead to inconsistent drug dosage. Although drug pumps can accurately control the dosage of each administration, they are not always compatible with the drug being delivered or the concentration being used. They also need to be placed surgically – which is more invasive to the animal, requiring an anesthetic event, and there is potential for post-surgical complications such as dehiscence. Subcutaneous injection, although more cumbersome, can ensure accurate dosage for each administration and maintain consistency in drug administration among different rats. At the same time, it has a lower cost and is suitable for conducting a large number of animal experiments.

This study applies repeated subcutaneous injection of scopolamine to establish a dry eye rat model. We analyze dry eye indicators such as corneal defects, tear secretion levels, and pathological morphology of the cornea, conjunctiva, and lacrimal gland. By combining drug concentration, pathological manifestations, and dry eye symptoms, we further elaborate on the dry eye rat model in detail, providing more accurate experimental data for the study of dry eye treatment and pathological mechanisms. We also describe the modeling process in detail for future researchers.

Protokół

All animal experiments performed following this protocol are performed under the approval of the Institutional Animal Care and Use Committee (IACUC).

1. Animal preparation

1. Prepare 12 healthy 6-week-old SPF Wistar female rats weighing 160 g ± 20 g.
2. Use a slit lamp and ophthalmoscope to examine the eye conditions of all rats, ensuring that there are no anterior segment or retinal diseases.
3. Raise all the rats for 1 week with sufficient food and water sources.
4. Randomly divided all rats into normal, scopolamine drug concentration 2.5 mg /mL, scopolamine drug concentration 5 mg/mL, and scopolamine drug concentration 7.5 mg/mL groups, with three animals in each group.

2. Solution preparation

1. Prepare scopolamine hydrobromide by dissolving it in 0.9% sodium chloride solution to make a solution with concentrations of 7.5 mg/mL, 5 mg/mL, and 2.5 mg/mL.
2. Prepare a 0.9% sodium chloride solution without scopolamine hydrobromide to be used as an injection for the control group of rats.

3. Equipment and material preparation

1. Prepare a small animal microscope.
2. Prepare materials for the experiment, including 1 mL disposable syringe with needle (26 G); fluorescein sodium ophthalmic strips; Schirmer tear test strip; absolute ethanol; 4% paraformaldehyde; xylene; neutral balsam; hematoxylin, eosin; and periodic acid-Schiff staining kit.

4. Subcutaneous injection

NOTE: This procedure requires assistance from a second person to help secure the rats.

1. Hold the rat's body steady and catch and stretch its left (or right) hind legs.
   NOTE: An assistant can help in holding the animal.
2. Clean injection site with alcohol.
3. Insert 1 mL disposable syringe with needle (26 G) at the base of skin fold between thumb and finger.
4. Aspirate the syringe by pulling back the syringe plunger. Any blood in the syringe indicates improper needle placement; remove and reposition the needle.
5. Administer 0.9% sodium chloride solution with or without scopolamine hydrobromide in a steady, fluid motion.
6. Inject all rats according to different concentrations, with 0.5 mL injected each time and four times daily (at 9:00, 12:00, 15:00, and 18:00) for a consecutive period of 19 days, alternating between left and right limbs.
   NOTE: The groups are named as follows:
   Group without scopolamine hydrobromide: 0 group (control)
   Group with scopolamine hydrobromide 2.5 mg/mL: 2.5 group
   Group with scopolamine hydrobromide 5 mg/mL: 5 group
   Group with scopolamine hydrobromide 7.5 mg/mL: 7.5 group
7. Return the animal to its cage and monitor breathing and behavior for 5-10 min.

5. Tear secretion test (Schirmer tear test, STT)

1. Create a modified filter paper strip for rats¹¹. Cut half of the filter paper strip used for humans along the centerline (1 mm × 15 mm), and trim the head of the strip to make it smooth.
   NOTE: Before conducting the tear secretion test, manually restrain the rat's body to prevent movement and ensure exposure of the rat's eyes.
2. Place the filter paper strip on the outer 1/3 of the lower eyelid conjunctival sac of the rat.
3. Time the test for 5 min. Control the closure of the rat's eyes throughout the procedure.
4. After measuring, use tweezers to clamp the filter paper strip into a microcentrifuge tube and record the tear volume by making a mark on the wall of the tube.
5. Measure tear secretion on day 0, day 1, day 3, day 5, day 7, day 11, day 15, and day 19.

6. Corneal fluorescein staining

1. Drop 0.5 µL of 0.5% fluorescein sodium solution into the inferior conjunctival sac of each rat.
2. Observe the cornea under blue light for 3 min after fluorescein instillation.
3. Record the fluorescence staining of each rat's cornea and observe whether there is a corneal defect.
4. Perform corneal fluorescein staining on day 0, day 1, day 3, day 5, day 7, day 11, day 15, and day 19.

7. Histological observation of conjunctival tissue

1. After completing the model development, anesthetize the rats deeply with an intraperitoneal injection of 0.4 mL/100 g of 10% aqueous chloral hydrate to alleviate the animals' tension. Then, euthanize the rats by cervical dislocation.
2. Take the bulbar conjunctiva from the same regions of each rat, with a size of approximately 2 mm x 2 mm.
3. Fix the tissues immediately in 4% paraformaldehyde for 24 h and embed in paraffin¹³.
4. Cut 5 µm thickness sections and stain with hematoxylin and eosin (HE)¹⁴ and periodic acid-Schiff (PAS) stain (follow manufacturer's instructions).

8. Histological observation of corneal and lacrimal gland tissue

1. After completing the model development, euthanize the rat as described in step 7.1.
2. Take the cornea on the right side of each rat and fix it immediately in 4% paraformaldehyde solution.
3. Cut the cephalic epidermis and subcutaneous tissue along the line connecting the ear and the outer corner of the eye, expand the incision to both sides and further isolate the yellowish extra orbital gland.
4. Thoroughly remove the rat's fur and separate the extraorbital gland with 0.9% sodium chloride solution.
5. Place the isolated extraorbital glands in 4% paraformaldehyde solution for 24 h and embed in paraffin.
6. Cut continuous sections of ~5 µm thickness and stain them with HE for corneal and extraorbital gland tissue specimens.

9. Statistical analysis

1. Use appropriate software for statistical analysis of the data.
   1. Perform one-way analysis of variance (ANOVA) to analyze the data and the least significant difference (LSD) test for comparison between groups. Set the statistical significance level at α = 0.05, with P < 0.05 indicating statistical significance.
      NOTE: SPSS 20 software was used for statistical analysis of the experimental data.

Wyniki

Schirmer I test, SIT I
The tear volume of the rats was measured on days 0, 3, 5, 7, 11, 15, and 19 after the start of the experiment. The experimental results showed that the tear secretion of the scopolamine group (2.5 group, 5 group, 7.5 group), compared with the control group (0 group), was significantly decreased, and the difference was statistically significant (P < 0.01). There was no statistical significance between the 2.5 group, 5 group, and 7.5 group (P > 0.05). There was no signi...

Dyskusje

Aqueous-deficient dry eye (ADDE) is an important type of dry eye, accounting for about 1/3 of the total dry eye population¹⁷, and the main cause of ADDE is lacrimal gland pathological damage and inflammation¹³. For this type of dry eye, the most common clinical treatment methods are artificial tears to alleviate symptoms or topical application of steroids or cyclosporine¹⁸, while there are few treatment options for damage to the lacrimal gland. There...

Materiały

Name	Company	Catalog Number	Comments
0.9% sodium chloride solution	SJZ No.4 Pharmaceutical	H13023201
4% paraformaldehyde	Wuhan Servicebio Technology Co., Ltd	G1113
Absolute ethanol	Sinopharm Chemical Reagent Co., Ltd.	10009218
Fluorescein sodium ophthalmic strips	Tianjin Yinuoxinkang Medical Device Tech Co., Ltd	YN-YG-I
Hematoxylin and eosin	Nanjing Jiancheng Bioengineering Institute	D006
Neutral balsam	Beijing Solarbio Science & Technology Co., Ltd.	G8590
Paraffin	Beijing Solarbio Science & Technology Co., Ltd.	YA0012
Periodic Acid-Schiff Staining Kit	Beyotime Biotechnology	C0142S
Schirmer tear test strips	Tianjin Yinuoxinkang Medical Device Tech Co., Ltd	YN-LZ-I
Scopolamine hydrobromide	Shanghai Macklin Biochemical Co., Ltd	S860151
Small animal microscope	Head Biotechnology Co,. Ltd	ZM191
Xylene	Sinopharm Chemical Reagent Co., Ltd.	10023418