Set up a quiet room with a controlled temperature of 21 degrees Celsius in a 12-hour light and 12-hour dark cycle. Gently place each mouse into a chamber made of PMMA placed on a five square millimeter mesh floor stand. Allow the mice to acclimate for 30 minutes before assessing baseline and experimental mechanical thresholds.
Then adjust the mesh floor stand to a comfortable height so that the mice can be assessed from all sides, and their abdomen and all four paws are easily reachable through the mesh floor openings. Cover the surface beneath the chambers with absorbent material to manage micturition and defecation. Ensure that there is ample arm space below the chambers while using the EVF apparatus.
After setting up the EVF apparatus for mechanical threshold assessments, conduct abdomen measurements by dividing the mouse abdomen into three virtual sections. To obtain baseline measurements, 48 hours before infection, place the mice in the chambers, and half an hour after setting up the EVF apparatus, stimulate the designated tissue by gradually elevating the probe with both hands. Now, apply gradual pressure to the tissue until the mouse displays nociceptive behavior.
To evaluate the right hind paw, watch for actions such as retraction, paw licking, or four-paw jumping. For visceral assessment, observe sudden abdominal retraction, immediate licking or scratching of the probed area, or four-paw jumping. Then save the recorded value into the designated electronic device with the EVF program or document it in a notebook.
Reset the display to zero. Probe each mouse in the adjacent chamber five times, calculate their individual average values and reassess the baseline measurements after 24 hours.
