This protocol outlines the steps to induce reliable and robust ocular inflammation using heat-killed mycobacteria in the mouse model. This primed mycobacterial uveitis model can help study the mechanisms of chronic immune mediated ocular inflammation that develops after a mycobacterial infection. This model of chronic uveitis does not require the use of specific mouse strains or immunization with ocular antigens.
This model facilitates the use of transgenic and gene knockout mouse lines in mechanistic studies of the pathogenesis of ocular inflammation. This PMU model will be helpful in studies testing new therapies for ocular inflammation or uveitis in humans. Treatments such as new eyedrops or systemically administered immune modulating agents could be tested for efficacy with this model.
This model will facilitate studying how heat-killed mycobacterial antigens alter the microenvironment of an immune privileged organ like the eye. These results could also provide insights on how other types of ocular infection impact the eye or how immune privileged sites, like the brain, respond to microbacterial antigens. To begin, un-clamp the body of the Sonicator converter unit and clean the probe with a 70%alcohol swab.
Then switch the Sonicator on and adjust the power setting to four by turning the power control knob. Next, to generate a fine suspension of the microbacterium tuberculosis H37Ra in PBS, immerse the probe's tip into the PBS-containing microbacterial powder. Sonicate the mixture on ice for 30 seconds, then pause for 30 seconds and repeat for a total of five minutes to fully disperse the powder into an even suspension without heating the liquid.
Next, add 2.5 milliliters of Freund's Incomplete Adjuvant to the mixture, and repeat the sonication process on ice until the emulsion forms a toothpaste-like consistency. For the subcutaneous injection, load 200 to 300 microliters of the microbacterial emulsion in a one milliliter syringe. Expel the air from the syringe and continue filling the syringe with intermittent inverting and tapping until filled.
Next, place the subcutaneous injections on either the dorsal surface of the hips or on the ventral surface of the legs proximal to the region of the inguinal lymph nodes of a 6 to 10 week old anesthetized C57 black 6J mouse. Carefully insert the needle, taking care not to penetrate the muscle, and inject 50 microliters of the emulsion into the subcutaneous space. Do not remove the needle immediately to allow the thick emulsion to be fully injected.
To prepare the antigen stock for the intravitreal injection, sonicate the H37Ra in PBS solution as demonstrated previously, then aliquot the solution in 100 microliter volumes and store at 20 degrees Celsius. 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's 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 one to two 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 one 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 10 seconds, remove the needle from the eye.
After anesthetizing the mouse, dilate the pupil with one drop of 2.5%phenylephrine, avoiding excess droplets entering the nose or mouth. After two to three minutes, dab off the excess liquid and cover the eye with 0.3%hypromellose gel. Next, wrap the mouse in a layer of surgical gauze to maintain body warmth, then place the mouse on the animal cassette and position the head with the bite bar.
To acquire the optical coherence tomography or OCT images, turn on the OCT imaging system and open the imaging software. For posterior chamber imaging, bring the OCT close to the eye's surface, taking care not to bring the surface of the lens in contact with the eye. Once the eye is correctly positioned, stop the fast scan, select the volume scan protocol, and activate the scan with the Aim option.
For posterior segment images, adjust until the optic nerve is centered in the horizontal B scan alignment image, and the retina is aligned with the vertical alignment axis. For anterior segment images, adjust the position to center the apex of the cornea in both the horizontal B-scan alignment image and vertical B-scan alignment image. Finally, click on Snapshot to capture the volume scan image, then click on save.
24 hours after intravitreal injection, inflammatory cells are seen in the aqueous and vitreous. Corneal edema, a hypopyon, and multiple free-floating inflammatory cells are seen in the anterior chamber, and vitritis in the posterior chamber. The degree of ocular inflammation can be scored on these OCT images.
Combined scores greater than zero but less than 2.5 represent mild inflammation. Scores greater than 2.5, but less than 4.5. represent moderate inflammation, and scores greater than 4.5 identify severe inflammation.
Inflammation scores can also be determined using five characteristics visible in hematoxylin and eosin stain sections. The severity score is determined by counting the number of inflammatory cells in the aqueous and vitreous. Brightfield longitudinal fundus imaging can also identify retinal and perivascular inflammation.
Severely inflamed eyes demonstrate multiple white infiltrates in the retina and vascular tortuosity on color fundus imaging, as well as dense vitritis and retinal edema on OCT on day two. Mildly inflamed eyes demonstrate fewer and more discreet linear lesions in the fundus, and a number of infiltrating cells in the vitreous space. Ensuring consistency during the subcutaneous and intravitreal injections and taking appropriate measures to prevent the development of infectious endophthalmitis are the key steps that help in successfully replicating this model.
In vivo bioluminescence imaging can be used to characterize inflammation while post-mortem assays, like multi-parameter, flow cytometric analysis can be performed to identify and quantify infiltrating immune cell type populations. Cytokine analysis, mRNA sequencing, and immunofluorescence imaging can be used to assess gene and protein expression patterns or identify retinal immune cell populations in uveitis. This protocol shows how to perform reproducible, aseptic, and minimally traumatic injections that will help future investigators access the intravitreal space in mice.
OCT will also play an important role in detecting ocular inflammation that could otherwise be missed by a visual examination of the eyes of small experimental animals.
