A Method to Induce Ocular Surface Inflammation in a Murine Model

The ocular surface system comprises the cornea, conjunctiva, and an associated gland network. A tear film protects the ocular surface.

The evaporation of the tear film's aqueous layer is inhibited by a lipid layer secreted by the meibomian glands. Ocular surface inflammation obstructs lipid secretion, leading to dry eye disease.

To induce ocular surface inflammation in a murine model, take a mouse and inject an immunogen solution intraperitoneally. The immunogen activates helper T cells, or Th cells, to elicit an immune response. The activated cells spread to the eyes through systemic circulation.

Post-incubation, perform an ocular surface challenge by applying the immunogen on the eye, generating an exaggerated immune response.

Antigen-presenting cells present the immunogen to the activated Th cells. Restimulation of the Th cells causes the release of cytokines and chemoattractants, leading to ocular surface inflammation.

The chemoattractants cause an influx of neutrophils. Binding to the cytokines activates a signaling cascade inside the neutrophils, generating reactive oxygen species or ROS. The generated ROS causes the release of antimicrobial proteins from cytoplasmic granules and chromatin decondensation.

The decondensed chromatin fibers and granular proteins form an extracellular web-like structure termed an aggregated neutrophil extracellular trap or aggNET. The excessive aggNET production clogs meibomian gland openings — preventing lipid secretion.

The murine model of ocular surface inflammation is ready for downstream analysis.

To perform intraperitoneal injection of the immunogen solution, hold the 7 to 9-weeks-old C57 Black 6 non-anesthetized female mouse softly by the tail while grasping the cage grid. Then, firmly hold the skin at 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 5 microliters of ovalbumin, or ova, or saline per eye of an anesthetized mouse, and wait for 5 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.