Safety assessment of temporal interference stimulation

Temporal interference (TI) stimulation is a promising non-invasive neuromodulation strategy that uses two high-frequency electric fields to generate a low-frequency amplitude-modulated envelope at their intersection, enabling targeting of deep brain regions. However, <i>in vivo</i> safety concerns remain regarding the impact of the low-frequency envelope applied to the brain. Therefore, the objective of this study was to systematically evaluate the acute thermal and cellular safety profile of TI using an invasive <i>in vivo</i> mouse model, and to compare its thermal effects with those of direct low-frequency stimulation using an <i>in vitro</i> egg-white model. In the egg-white model, no protein coagulation was observed with TI stimulation (10 mA at 1,000 Hz and 1,005 Hz for 20 min), which generated a 5 Hz envelope. In contrast, conventional 5 Hz alternating current stimulation (tACS) at 10 mA induced localized coagulation. In the mouse model, intracranial TI stimulation (2 mA at 1,000 Hz and 1,005 Hz for 20 min) targeting the hippocampus resulted in a mild and stable temperature increase of ∼0.7 °C. Histological analysis revealed a localized increase in astrocyte activation (GFAP) in the stratum lacunosum-moleculare (SLM) compared to other hippocampal subfields. No significant expression difference was observed in the hippocampus for the heat stress marker (HSP70) or the inflammatory marker (iNOS). These findings suggest that TI has a favorable short-term safety profile, with minimal thermal effects and no widespread inflammatory response.