tFUS safety evaluation in female HbSS-BERK mice. (A) The single- and multisession tFUS treatment conditions used in safety study. (B-C) The effect of tFUS on the motor coordination and balance was investigated in rotarod testing. After 60 minutes of single-session tFUS stimulation with a PRF of 40 Hz to S1HL (n = 11) or thalamus (n = 11) and after 60 minutes of multisession tFUS with a PRF of 40 Hz to S1HL (n = 4) or insula (n = 6) for 14 days with 1 hour per day, no significant alteration was observed in the averaged latency to fall compared with prestimulation baseline as well as control groups. In addition, there was no significant difference of control groups from their baseline values using t test with Wilcoxon matched–pairs signed rank test and 1-way ANOVA with Kruskal-Wallis test. (D) Double-blind histological analysis using hematoxylin and eosin (H&E) and TUNEL stains showed that multisession tFUS stimulation to the left S1HL or insula had no adverse impact on tissue structural integrity and nuclear morphology within the treated brain region compared with contralateral brain regions and sham treatment. Scale bars, 50 μm. ns, not significant; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.