This article describes a method for in vivo imaging of inflammation in mice with experimental autoimmune encephalomyelitis (EAE). The technique utilizes bioluminescent substrates to visualize inflammation in the optic nerve and brain.
Begin with an anesthetized mouse with experimental autoimmune encephalomyelitis or EAE. This condition causes inflammation in the eyes and the brain.
Place the mouse in an imaging apparatus, aligning the upper back at the center.
Cover the lower portion of the mouse with a black cloth to prevent signal interference from this region.
Use imaging software to record the mouse position.
Take a syringe filled with a reagent containing the bioluminescent substrate and inject it intraperitoneally into the mouse.
The reagent circulates and enters the inflammation site, where the substrate interacts with inflammation-specific enzymes.
The enzyme-substrate reaction produces a bioluminescent signal.
Capture images at regular time intervals to monitor this signal.
The optic nerve lies near the surface, making bioluminescence around the eyes prominent. In contrast, signals from the brain are weak as the skull absorbs the emitted light.
In the image, blue indicates mild inflammation, and red indicates severe inflammation.
To perform in vivo imaging, keep the mice under 2 to 2.5% isoflurane anesthesia during all imaging procedures. Position two mice next to each other in the apparatus, and position the upper spines of the mice in the center.
Next, shield the site of immunization with black cloth. Open the software for image capture and initialize the camera. Then, take a baseline image to assess the correct positioning of the mice.
Subsequently, use B focus with a 6.5 centimeter distance to the camera for capturing all the images. Afterward, mix the ready-to-use chemiluminescent reagent at 40 milligrams per milliliter before filling the syringe. Then, inject 100 microliters of it in the mice intraperitoneally and capture the bioluminescence images 5, 10, and 15 minutes after injection.
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