To begin, confirm the anesthetization of the mouse by assessing the absence of a response to a toe pinch. Secure the mouse into the inner animal holder with its head and upper hind shoulders exposed through the rectangular opening while the dorsum and lower hind limbs remain shielded. Apply surgical tape across the upper hind shoulders to secure the mouse.
Insert the inner animal holder into the outer holder. After opening the secondary anesthesia line inside the animal holder, seal off the end of the holder to prevent the anesthetic from leaking out of the tube and the mouse from moving during the experiment. Align the outer animal holder's four millimeter circular aperture directly over the mouse's left eye.
Position the animal holder on the XY table using the control knobs, so that its aperture aligns with the barrel of the paintball gun, and the external surface is five millimeters from the end of the barrel. After calibration, initiate the over-pressure air sequence to deliver two bursts of 15 PSI at an interval of 0.5 seconds. For the sham group mouse, turn the animal holder away from the barrel, block the air with a cardboard shield and expose it to the noise of the over-pressure air.
Healthy densely packed axons were observed in the sham group. In contrast, the ITON group exhibited signs of axon degeneration, including irregularities in axon shape and myelin sheath breakdown. The total number of axons significantly decreased in the ITON group compared to the sham group.
A significant increase in degenerative profiles was detected in the ITON group compared to the sham group. Increased microglia proliferation was observed in the retina of ITON mice, with microglia extending into the outer nuclear layer. Compared to the sham group, where microglia remained in their typical retinal layers.
Intact synaptic connections between rod bipolar cells and photoreceptors were noted in both ITON and sham mice, with no detected synaptopathy.
