Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

We are dedicated to establishing a reliable model of dry eye syndrome. We have successfully replicated the pathological state of aqueous deficiency dry eyes by removing ELG and ILG from eyes. Due to the deep anatomical location of ILG, the combined excision of ELG and ILG is more challenging and invasive.

It is also able to cause bleeding during the operation, which puts a high demand on our operating ability. Our model has successfully induced significant aqueous humor deficient dry eye with minimal damage to the animal, and significant simplification of the surgical procedure. The constructed mouse model is highly similar to severe dry eye syndrome, such as Sjogren's syndrome and Stevens-Johnson syndrome, which provides strong support for related research.

The method avoids systematic injection effects, addresses the poor durability of the local injection effect, overcomes shortcomings of the dry eye model, and simplifies the experimental process by eliminating the need for a dedicated animal environment. To begin, place the anesthetized mouse in the lateral decubitus position, and expose the surgical area under an operating microscope. Using toothed forceps, clamp the skin on the right side of the mouse's face.

Make an incision at the midpoint of the line, intersecting the external oracle and the mandibular line to the inner corner of the eye. Then expose the muscle tissue and carefully remove the ELG located on the muscle. Now extend the skin incision to the inner corner of the eye.

Bluntly separate the muscle and locate the light red gland beneath the muscle. Then peel off and remove the ILG. Suture the incision using 5-0 sutures with a needle holder, ophthalmic surgical scissors, and forceps.

Apply ofloxacin ointment at the end of the operation to prevent infection. After removing the unilateral ELG and ILG, place the mice in an environment with a rhythmic 12-hour light-dark cycle and free access to food and water. Take a well-packaged phenol red thread for tear measurement.

After anesthetizing the aqueous deficiency dry eye mouse, gently pull down the lower eyelid of the eye to be measured. Position the top of the phenol red thread into the inner and outer thirds of the lower eyelid and initiate the timer promptly. After the allotted time, carefully retract the lower eyelid and remove the phenol red thread downward with caution.

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. Employ an electronic stopwatch to accurately record the duration of the test. To prepare tissue slices for staining, take the eyeballs extracted from the euthanized mouse.

Then embed the eyeballs in an optimal cutting temperature compound and utilize a frozen microtome to obtain freezing tissue slices of five to seven micrometers thickness. For corneal mRNA extraction, use scissors and forceps to cut the eyeball from the posterior pole under the microscope. Separate the lens and clean up the excess sclera and iris, but leave the 0.5 millimeter white sclera and the entire cornea.

Following a two-week period post tear gland resection, tear secretion in the dry eye mice notably decreased compared to the normal group. Squamous metaplasia and accumulation of inflammatory cells were observed in the corneal epithelium and stromal layer of the dry eye group. The expression of keratin 12 was significantly lower in the corneal epithelial cells of the dry eye group compared to the normal group.

Pax6 expression was significantly reduced in the corneal epithelial cells of the dry eye group compared to the normal group. Expression of Sprr1b, a marker of abnormally differentiated corneal epithelial cells, was significantly higher in the dry eye group than in the normal group.