We tried to establish an optimum FVIO-mediated stimulation method which satisfies the neuromodulation requirement while resulting in the least tissue damage on the brain region[47]. Therefore, we further evaluated the biosafety of FVIO-mediated magnetothermal neurostimulation in mice, which is vital for brain safety[53-54]. Representative hematoxylin and eosin (H&E)-stained sections from FVIO (0.05 μg)-treated mice exhibited no histopathological alterations in the CeA region (Figure 5a). In contrast, SPIO treatment at the minimum dose of 0.80 μg caused significant histopathological changes in the CeA regions of the mice, including vacuolar degeneration and nuclear chromatin condensation and fragmentation. After magnetothermal treatment, the SPIOs induced neuronal loss in the CeA regions of the mice, as shown by Nissl staining. More shrunken neurons with pyknotic nuclei were found in the SPIO-treated mice than in the FVIO-treated mice (Figure 5a). We further assessed inflammation in the CeA regions of the mice after magnetothemal neurostimulation using immunohistochemical analysis (Figure 5b). The expression levels of proinflammatory cytokines (interleukin-6 (IL-6), TNF-α, and IL-1β) in FVIO-treated mice were significantly lower than those in SPIO-treated mice (Figure 5, c to e). This was ascribed to the high dose of SPIOs (0.8 μg per CeA region) needed, which caused unfavorable heat damage in the CeA region. These results imply that FVIO-mediated magnetothermal neurostimulation is much safer than SPIO-based treatment.