Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis

All procedures performed were approved locally by the Animal Care and Use Committee at the University of Washington (animal study protocol # 4481-02) or in concordance with the United Kingdom Home Office license (PPL 30/3281) and the University of Bristol Ethical Review Group. Experiments conducted at both institutions were concordant with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research. PMU was generated in 6-10-week-old C57BL/6J mice; all mice weighed at least 18 g at the time of uveitis induction and were confirmed negative for the rd8 mutation of the Crb1 gene²⁹. Mice were maintained with standard chow and medicated water (acetaminophen 200-300 mg/kg/day) ad libitum under specific pathogen-free conditions. Animal euthanasia was performed using a standard carbon dioxide inhalation method³⁰.

1. Antigen preparation for subcutaneous injection

1. Perform all procedures in this section inside a chemical fume hood to prevent inhalation or skin contact with the Mtb H37Ra powder. Handle according to your institutional policies for Complete Freund's Adjuvant (typically BSL-1). This includes using chemical-resistant gloves, safety glasses, and protective work clothing (lab coat).
2. Use a good sterile technique to prevent contamination of reagents that will be introduced into experimental animals.
3. Make the Mtb suspension in PBS by mixing 5 mg of lyophilized, heat-killed M. tuberculosis H37Ra powder with 2.5 mL of cold PBS in a 5 mL microcentrifuge tube. Vortex once for 30 s and then place on ice.
4. To generate a fine suspension of the H37Ra in PBS, sonicate the suspension on ice for 5 min.
   1. Unclamp the body of the converter unit and clean the probe with a 70% (v/v) alcohol swab.
   2. Switch on the sonicator, adjust the power setting to 4 by turning the power control knob and immerse the probe's tip into the PBS-containing mycobacterial powder. Ensure that the probe tip is immersed to at least half the depth of the sample and that the probe tip is not touching the wall of the microcentrifuge tube.
5. Sonicate the mixture on ice for 30 s, pause 30 s and repeat for a total of 5 min to fully disperse the powder into an even suspension without heating the liquid.
6. Add 2.5 mL of Freund's Incomplete Adjuvant to the mixture and repeat the sonication process on ice until the emulsion forms a toothpaste-like consistency.
7. Set the power to 0 using the control knob and turn off the unit to end the sonication. Remove the tip from the suspension and wipe the probe with an alcohol swab.
8. Store the antigen emulsion at 4 °C. Making batches of the emulsion will help ensure consistency across experiments. The emulsion can be stored at 4 °C for up to 3 months.

2. Subcutaneous injection

1. Perform subcutaneous injection a week prior to the intravitreal injection (designated as day -7).
2. Load a 1 mL syringe (no needle attached) with the mycobacterial emulsion. Due to the viscosity and opacity of the emulsion, difficult-to-see air bubbles can fill the syringe.
   1. To prevent airbubbles in the syringe, after loading 0.2-0.3 mL of emulsion, invert the syringe (tip facing up) and gently tap the syringe repeatedly on the edge of a counter to bring the bubbles to the surface.
3. Expel the air from the syringe and continue filling the syringe. Invert and intermittently tap until filled.
4. Place a 25 G needle on the syringe and advance the emulsion to fill the needle. Store the syringe on ice until used.
5. To perform the subcutaneous injection safely, either anesthetize the mouse or utilize humane restraint methods that allow easy access to the animal hindquarters³¹.
6. To anesthetize for subcutaneous injection, place the animal in an isoflurane induction chamber (3%-4% for induction and 1%-3% for maintenance). Once anesthetized, ensure that the mouse has a slow respiratory rate and exhibits no signs of respiratory distress.
7. 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.
   1. Carefully insert the needle to prevent injecting into the muscle. Inject 0.05 mL of the Mtb emulsion into the subcutaneous space. Do not remove the needle immediately in order to allow the thick emulsion to be fully injected.
   2. Repeat the injection on both left and right sides for a total of 0.1 mL per animal.
8. If anesthetized, place the mouse on a warm heating pad until complete recovery. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency.
9. Return the mouse to its cage upon complete recovery and label the cage card with the date of subcutaneous injection.
10. Provide analgesia with oral acetaminophen (200 mg/kg/day), but not NSAIDs as anti-inflammatory agents can impact the induction of uveitis.

3. Antigen stock preparation for intravitreal injection

1. Perform all procedures in this section under appropriate sterile conditions to prevent contamination of the intravitreal Mtb suspension.
2. Make the intravitreal suspensions.
   1. For induction of mild to moderate panuveitis, make the intravitreal suspension at a 5 mg/mL concentration by adding 5 mg of the mycobacteria extract to 1 mL of 1x PBS.
   2. For induction of moderate to severe panuveitis, make the intravitreal suspension at a 10 mg/mL concentration by adding 10 mg of the mycobacteria extract to 1 mL of 1x PBS.
3. Vortex once for 30 s and then place on ice.
4. To generate a fine suspension of the H37Ra in PBS, sonicate the suspension on ice for 10 min as described in step 1.4. Aliquot this stock solution in 100 µL volumes and store at -20 °C.
5. Prior to use, thaw at room temperature and vortex on high for 1 min. Keep the aliquots on ice while transport to the animal facility.

4. Intravitreal injection procedure on day 0

1. Animal preparation
   1. Wear fitted examination gloves, place the mouse on a weighing balance to obtain its weight in grams.
   2. Give an intraperitoneal injection of 0.02 mL/g bodyweight of a solution containing 100 mg/mL Ketamine and 20 mg/mL Xylazine mixed with sterile water to anesthetize the animal. An alternative approach includes induction using ~1.5% isoflurane (inhaled).
   3. Wait approximately 2 min for the mouse to fall asleep, and then place the mouse in a warming box and cover the lid. Perform pain reflex tests like ear, toe, and tail pinch to assess the depth of anesthesia for the procedure³².
   4. Once asleep, anesthetize the cornea with 1 drop of 0.5% (v/v) tetracaine. Avoid getting tetracaine near the nose or mouth of the mouse. After 10 s, dab off the excess liquid.
      NOTE: It has been observed that iris dilation and anterior chamber (AC) visualization are improved when topical anesthesia is administered, possibly due to improved corneal reflex suppression with the combined systemic and topical anesthesia. However, this step could be omitted if desired.
   5. Dilate the pupil with 1 drop of 2.5% (v/v) phenylephrine. Use caution to avoid any excess droplets that might enter the nose or mouth. After 2-3 min, dab off the excess liquid.
   6. To decrease the risk of endophthalmitis, add 1 drop of 5% betadine to the eye surface and surrounding hair. Leave on the eye for 2-3 min.
      NOTE: Perform all procedures in this section under appropriate sterile conditions to prevent endophthalmitis.
   7. Remove betadine and cover the eye with hypromellose (0.3%) or carbomer eye gel 0.2% w/w) to prevent dryness under anesthesia. This will also help prevent cataract formation.
2. Setting up the microinjection system
   1. Perform the intravitreal injection using a micropump connected to a microsyringe pump controller and an injection syringe (Figure 1A-C). Alternatively, inject with a 33 G needle attached to a Hamilton syringe as described in subsection 4.4.
   2. Connect a 34 G needle to the injection holder to assemble the injector. Loosen the silver screw cap at the front end of the injection holder and slide the needle into the body of the holder about halfway. Tighten the silver screw cap finger tight.
   3. Connect the tubing to the injection holder as mentioned in steps 4.2.4-4.2.5.
   4. To insert the tubing on the injection holder, loosen the plastic screw on the back end of the holder, slide the tubing through the gasket inside and tighten the screw.
   5. Maintain a slight gap with the end of the tubing to prevent tubing damage during the injection. Refer to Figure 1C.
   6. Thaw a 100 µL aliquot of the mycobacterium stock suspension.
   7. Add 3 µL of a 1% fluorescein sodium (AK-Fluor) solution and vortex well.
   8. Load a 10 µL syringe with the antigen and fluorescein mix without including any air bubbles.
   9. Remove the loading needle from the syringe, and slide the tubing through the silver screw cap gasket until the tip reaches the zero mark in the syringe body.
   10. Once the tip of the tubing is correctly aligned to the desired position, tighten the screw cap finger tight.
   11. Flush the solution in the syringe through the injection tubing to fully load the system. Then repeat steps 4.2.8-4.2.11 to reload the syringe for injection.
   12. To install the loaded syringe on the micropump, press the clamp release button at the end of the micro-pump to open the syringe clamps.
   13. Position the cap of the plunger into the plunger cap holder at the rear end of the micropump.
   14. Then slide the syringe collar onto the collar stop and the syringe body into the syringe clamp.
   15. Release the clamp button and tighten the plunger retaining screw. Refer to Figure 1D.
   16. Slide the injection holder and the needle through the o-clamp on the stereotactic injection apparatus. This is a custom platform; alternatively, the syringe can be held and positioned manually.
   17. Set the infusion volume and the rate of infusion volume on the microsyringe pump controller to inject 500 nL per cycle at a rate of 40 nL/s, respectively.
       NOTE: Faster injection rates can be used, however, more reflux may be experienced before needle repositioning can be achieved.
   18. Test the system to ensure correct functioning prior to performing an intravitreal injection.
       NOTE: When the injection system is functioning correctly, activation of an injection cycle using the foot pedal or the control pad will produce visible movement of the plunger cap holder and a small droplet of greenish liquid will be seen at the tip of the needle. In the case that no liquid is produced, activate additional cycles or flush and reload the syringe.
   19. Prior to injecting the eye, gently wipe the needle with a 95% ethanol pad.
3. Intravitreal injection procedure
   1. The mouse is placed on a stereotaxic apparatus to perform the injection procedure.
   2. Keep the stage/platform on which the mouse rests warm by attaching 2-3 paper towels on its surface.
   3. Place the mouse in a prone position on the platform. Use the right and left ear bars to gently fix the animal's head. Refer to Figure 1E.
   4. Position the mouse and orient under the scope so that the superior nasal aspect of the right eye is visible.
   5. Use a 30 G needle to displace the eyelashes and expose the sclera. Visualize the limbus and the radial blood vessel.
   6. Use a sterile 30 G needle to make a guide hole in the sclera 1-2 mm posterior to the limbus.
   7. Insert the 34 G 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.
   8. Using the microsyringe pump controller, carefully inject 1 µL of the Mtb extract into the vitreous cavity. In case of consistent reflux, increase the injection volume to 1.5 µL to ensure adequate dose delivery.
      NOTE: For sham controls, inject 1 µL of PBS into the eye of the animal.
   9. Verify intravitreal placement by visualization of a greenish reflex in the eye. Refer to Figure 1F.
   10. After 10 s, withdraw the needle from the eye. Note any reflux.
   11. Remove the mouse from the platform, place 0.3% hypromellose or 0.2% w/w carbomer eye ointment on both eyes for corneal protection, and move to the recovery warming box.
   12. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return to the company of other animals until fully recovered.
   13. When the mouse is fully awake, return to the cage and add acetaminophen (200-300 mg/kg/day) medicated water bottle. Label the cage card with the date of IVT injection.
   14. Intravitreal injections are generally well tolerated. Clinical signs that may indicate pain and the need for removal from the study include periocular alopecia (indicative of self-trauma), corneal ulceration, weight loss, and hunched posture.
4. Alternative method for intravitreal injection
   ​NOTE: This procedure is performed using an operating microscope and a 33 G needle on a microsyringe.
   1. Proptose the eye and hold it in position with a pair of forceps.
   2. Then apply carbomer eye gel 0.2 % w/w or 0.3% hypromellose eye gel and place a circular coverslip (7 mm diameter) over the eye.
   3. Mount a 33 G hypodermic needle on a 5 µL Hamilton syringe and insert it approximately 2 mm circumferential to the corneal limbus with a ~45° injection angle.
   4. Guide the needle bevel into the vitreous, stopping between the lens and the optic disc (from the relative viewpoint of the surgeon, this is above/covering the optic disc – approximately 1.5 mm from the site of insertion), and inject 2 µL of Mtb (at 2.5 ug/µL in PBS) slowly.
   5. Hold the needle in place briefly (to reduce the amount of reflux of injectate) and then remove it.
   6. Post-injection, reposit the globe by releasing the forceps. A drop of 1% w/w chloramphenicol ointment can be administered to the eye at this time to provide additional protection from post-injection endophthalmitis. 
   7. Post-injection, move to the recovery warming box as mentioned in steps 4.3.11-4.3.13.

5. OCT imaging to detect and quantify uveitis

1. Animal preparation
   1. Anesthetize the mouse as described in steps 4.1.2-4.1.4
   2. Dilate the pupil with 1 drop of 2.5% phenylephrine. Use caution to avoid any excess droplets that might enter the nose or mouth. After 2-3 mins, dab off the excess liquid.
   3. Place 0.3% hypromellose or 0.2% w/w carbomer gel on the eye to prevent dryness while under anesthesia. This will also help to prevent cataract formation.
   4. Wrap the mouse in a layer of surgical gauze to maintain body warmth and place it on the animal cassette. Position the head with the bite bar.
2. Acquire the OCT images of the anterior and posterior chambers.
   NOTE: If obtaining anterior and posterior chamber images, obtain the posterior chamber (PC) images first to prevent image degradation following cataract formation. Cataract formation can be prevented with frequent lubrication and applying 0.3% hypromellose or 0.2% w/w carbomer eye gel. For extended imaging (>10 min), keeping the mouse warm (through the use of a heat pad) also helps.
   1. After turning on the OCT imaging system, secure the correct imaging lens and adjust the reference arm position as needed.
   2. Open the imaging software, create the unique mouse ID and begin imaging per OCT manufacturer's protocol.
   3. Using the Free Run option with the fast scan protocol, position the eye with the optic nerve centered on posterior chamber images or the apex of the cornea on anterior chamber images.
      NOTE: Table 1 contains imaging protocol parameters for two commercially available small animal imaging systems. Refer to the Table of Materials for product specifications.
   4. For posterior chamber imaging, bring the OCT close to the surface of the eye. Use caution to avoid bringing the surface of the lens in contact with the eye.
   5. Once the eye is correctly positioned, stop the fast scan and select the volume scan protocol, and activate the scan with the Aim option.
   6. For posterior segment images, adjust untilthe optic nerve is centered in the Horizontal B-Scan Alignment image and that the retina is aligned with the Vertical Alignment axis
   7. For anterior segment images, adjust the position to center the apex of the cornea in both the Horizontal B-Scan Alignment image and Vertical Alignment B-Scan Alignment image. The presence of a reflection artifact in both images will confirm proper alignment. Then shift the horizontal image enough to remove the reflection artifact. Refer to Figure 2.
   8. Click on Snapshot to capture the volume scan image and then click on Save.
   9. Next, obtain the averaged central line scan. Open the scan protocol and click on Aim followed by Snapshot. Right-click on the same panel and then click on Average.
   10. Repeat steps 5.2.1-5.2.9 for each eye with both lenses.
   11. After all images are collected, remove the mouse from the cassette and provide corneal protection during recovery as listed in steps 4.3.11-4.3.13.

6. Scoring inflammation by OCT

1. Score the OCT images with the help of graders who are masked to the treatment condition.
   NOTE: For PMU in the mouse model, the scoring system provided in Table 2 is recommended.
2. If both anterior chamber (AC) and posterior chamber (PC) images were obtained, combine these scores to obtain the final score for each eye.
   ​NOTE: Anterior chamber inflammation resolves before posterior chamber inflammation.

7. Scoring inflammation by post-mortem histology

1. At the end of the experiment, collect individual eyes by enucleation, fix in 4% formaldehyde overnight, and proceed for paraffin embedding, sectioning, and H&E staining³³.
   NOTE: Multiple 4-8 µm sections along the pupillary-optic nerve axis are recommended.
   NOTE: Three sections per eye are scored by a masked grader using the scoring system provided in Table 3, and the average score of the three sections is reported as the final histology inflammation score.