This article describes a model for post-infectious uveitis induced by Mycobacterium tuberculosis in mice. The methodology involves immunization and intravitreal injection to study the inflammatory response in the eye.
Chronic eye inflammation, or uveitis, following a systemic bacterial infection, is termed post-infectious uveitis.
To generate a post-infectious uveitis model, take an anesthetized mouse previously immunized with heat-killed Mycobacterium tuberculosis or Mtb. Priming — prior exposure to the immunogen — generates circulating Mtb-specific T cells.
Inject the same immunogen into the vitreous cavity of the eye. Mycobacteria diffuse through the vitreous humor, spreading across the eye. The ocular-blood barriers restrict the entry of immune cells into the eye — making it an immune-privileged organ.
Tissue-resident antigen-presenting cells or APCs — in the perivascular region — encounter the diffused immunogen. Upon internalizing the immunogen, APCs process the Mtb antigen and display it on the surface via the major histocompatibility complex or MHC.
Activated APCs also release pro-inflammatory cytokines that cause permeability of the endothelial tight junction barrier in the blood vessel. Leukocytes, including neutrophils and Mtb-specific T cells, extravasate into the perivascular space.
Mtb-specific T cells bind to its antigen on APCs and release pro-inflammatory cytokines, which induce neutrophils to produce reactive oxygen species, or ROS, and cause degranulation.
Released granular proteases and ROS cause degradation of tissue parenchyma and breakdown of the ocular-blood barrier — exacerbating the inflammation.
The infection model is ready for downstream analysis.
On the day of the injection, after confirming the depth of general anesthesia, anesthetize the cornea with one drop of 0.5% tetracaine, and dilate the pupil with one drop of 2.5% phenylephrine. After dabbing off excess liquid, add one drop of 5% betadine to the eye surface, and surrounding hair to decrease the risk of endophthalmitis.
After two to three minutes, remove the betadine, and cover the eye with 0.3% hypromellose gel to prevent dryness under anesthesia and cataract formation. Next, load a 10-microliter syringe with the antigen and fluorescein mix. Then, place the mouse in a prone position on the platform and use the right and left ear bars to fix the mouse head.
Position and orient the mouse under the scope so that the superior nasal aspect of the right eye is visible. Then, using a 30-gauge needle, displace the eyelashes, expose the sclera, and visualize the limbus and the radial blood vessel.
Next, using a sterile 30-gauge needle make a guide hole in the sclera 1 to 2 millimeter posterior to the limbus. Then, insert the 34-gauge needle attached to the injection holder into the eye through the guide hole at an angle that will avoid the lens, but place the needle tip into the vitreous cavity.
Using a micro syringe pump controller, carefully inject 1 microliter of the M tuberculosis extract into the vitreous cavity. In case of consistent reflux, increase the injection volume to 1.5 microliters to ensure adequate dose delivery. Confirm the intravitreal placement by visualizing a greenish reflex in the eye, and after ten seconds, remove the needle from the eye.
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