Induction of Ocular Surface Inflammation and Collection of Involved Tissues

This protocol sequentially describes the steps involved in the induction of ocular surface inflammation and the collection of associated affected organs involved in Meibomian gland dysfunction. This new model of MGD enables the investigation of innate immune aspect of the disease, particularly the formation of neutrophil extracellular traps occluding the Meibomian gland. The technique exhibited in the protocol can be extended to introduce topical therapeutic interventions in a pre-clinical stage.

Moreover, the collection of ocular exudates and organs reflect the efficacy of the treatment. To perform intraperitoneal injection of the immunogen solution, hold the seven to nine weeks old C57 black six non anesthetized female mouse softly by the tail while grasping the cage grid. Then firmly hold the skin of the back and the neck region between the thumb and index finger and fix the tail and lower limbs between the ring and little finger against the palm.

Keeping the mouse fixed with its head downward, inject 100 microliters of the prepared immunogen solution in the right or left quadrant of the lower abdominal cavity. To perform an ocular surface challenge after immunization, apply five microliters of ovalbumin or ova or saline per eye of an anesthetized mouse, and wait for five minutes to get the drop absorbed by the eye. Next, collect the ocular exudates by applying 50 microliters of sterile saline to the eye immediately after the challenge.

For the excision of ocular surface tissues, place the mouse on an even surface and disinfect the orbital area around the eye with a 70%ethanol swab. To form an incision around the eye, make an incision between the ear and the retroorbital sinus and along the surface above the squamous bone vertically, extending the incision horizontally below the lower eyelid along the maxillary bone and above the upper eyelid along the frontal bone. Carefully hold the dissected tissue around the eye using curved forceps, and pull the tissue and eyeball out.

Place the excised organs in sterile PBS. Then using a scalpel under the stereo microscope, trim the excess facial muscle tissue around the excised upper eyelids. To collect the conjunctiva place the upper eyelid on a dry Petri dish.

Then using fine tweezers and a scalpel peel the whitish mucus layer gently out of the inner surface of the eyelid. Next to dissect the cornea, place the eye globe on a new dry Petri dish on top of dry ice. After three minutes, place the dish onto the bench surface in a stable position.

Use fine, sharp scissors to make a small incision at the border of the cornea next to the limbus. Continue the incision with the scalpel around the eye globe, separating the sclera from the cornea. Then remove the iris and lens remnants from the cornea's backside by generously flushing with saline solution.

To assess the Meibomian gland and its orifices, place the excised eyelids in an upright position under the stereo microscope. Then according to the camera exposure time and ISO settings, capture images under white light epi-illumination. To assess the Meibomian gland area, place the excised eyelids in a horizontal position and turn on the backlight of the stereo microscope equipped with an infrared camera.

Capture images by adjusting the exposure time according to the camera ISO. Saline administered mice showed healthy ocular surfaces with eyes wide open. In comparison, ova application induces severe ocular surface inflammation, the narrow opening of the eyes, and signs of chemosis.

The excised eyelids showed large occlusions plugging the orifices of the Meibomian gland and edema, in contrast to the healthy eyelids showing small plugs of the gland lining the eyelid. Further examination of the gland's infrared transillumination reveals the racemic appearance of the acini of the Meibomian gland. The eyelids of saline challenged mice showed the round acini.

In comparison, ova applications induced the destruction and loss of some glands with the allergic eye disease or AED. The histological analysis of the eyelids displayed dilated Meibomian glands compared to mice administered with only saline. Incubating the eyeball on dry ice allowed incision at the cornea next to the limbus and separation of the cornea from the sclera.

This study facilitated the collection of the cornea. In addition, ova administration inflicted severe inflammation to the conjunctiva with a hyperemic appearance. Elevated levels of the chemokine CXCL-1 facilitate neutrophil extravasation, phagocytosis, and degranulation, and interleukin-6 mediating the acute phase response and neutrophil infiltration were observed in the supernatants of AED mice.

On the other hand, the concentration of interleukin-10 showed no significant changes in both naive and AED mice. This new model enables the development and the evaluation of efficacy of drugs that target the innate immune pathways, and is of great help in data mining the way of administration of anti-inflammatory agents. After appropriate collection of organs, highly multiplex methods like single cell sequencing and imaging mass cytometry are compatible with this protocol.