Subcutaneous Injection of Bacterial Antigen to Induce Systemic Inflammation in a Murine Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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 Mycobacterium tuberculosis, 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 phosphate-buffered saline (PBS) by mixing 5 mg of lyophilized, heat-killed Mycobacterium tuberculosis H37Ra powder with 2.5 mL of cold PBS in a 5 mL microcentrifuge tube. Vortex once for 30 seconds 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 seconds, pause for 30 seconds, 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 air bubbles 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's hindquarters.
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 Non-steroidal anti-inflammatory drugs (NSAIDs) as anti-inflammatory agents can impact the induction of uveitis.