One month after LISW exposure, conduct a pathological examination of the mouse's cochlea. Start hemoperfusion with lactated Ringer's solution, followed by transcardiac perfusion with 4%paraformaldehyde. After decapitation of the mouse, remove the cochlea and perfuse directly with 4%paraformaldehyde at four degrees Celsius overnight.
After acquiring the entire image of the cochlea, using ImageJ software, compute the cochlear frequency map to precisely localize the specific cochlear regions at different frequencies. Using the given formula, calculate the survival rate of the hair cells at each frequency. After acquiring high-resolution Z-stack images of the inner hair cells, calculate the number of synapses.
Visualize the hematoxylin and eosin-stained cochlea sections and count the number of spiral ganglion neurons in the middle turn of the Rosenthal's canal to calculate the spiral ganglion neuron survival per control. Compared to the control, fluorescent immunostaining showed intact cochlear structures with no significant loss of hair cells or nerve fibers across all groups. However, quantitative assessments indicated that the survival of outer hair cells was significantly lower in the 2.5 joules per square centimeter group.
Meanwhile, inner hair cell survival was consistent among groups. Synaptic ribbon counts were notably reduced in higher-frequency groups. This was paralleled by a decrease in spiral ganglion neuron density in the 2.5 joules per square centimeter group, indicating that cochlear degeneration is dependent on LISW energy exposure.
