Our laboratory explores the mechanisms controlling secondary degeneration after ITON, indirect traumatic optic neuropathy, with the goal of developing rational therapies for patients. Currently, the devices to induce CNS injury in the field include controlled cortical impact devices, weight drop models, and compression air-driven shock tubes. We have found that the duration of the interblast exposure interval contributes significantly to the amount of axon degeneration, and that injury to the optic nerve, similar to injury to the brain, significantly elevates the amount of reactive oxygen species, and that contributes to the secondary axon degeneration.
This protocol addresses the need for a device that offers precise control over injury location and severity, while maintaining clinical relevance for closed system injuries. It also offers a way to study the short interblast injury intervals of certain occupational environments. It's also cost-effective, easy to set up and use.
So while other devices also offer customization, this closed system injury device stands out for its ease of set up and use. And this protocol provides a detailed framework simplifying this process, enabling other researchers to quickly adapt and set up the device for their own specific experimental goals and needs. 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 it's 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 raw bipolar cells and photoreceptors were noted in both ITON and Sham mice, with no detected synaptopathy.
