Background: Neuromodulation by transcranial focused ultrasound (FUS) offers the potential to non-invasively treat specific brain regions, with treatment location verified by magnetic resonance acoustic radiation force imaging (MR-ARFI).Objective: To investigate the safety of these methods prior to widespread clinical use, we report histologic findings in two large animal models following FUS neuromodulation and MR-ARFI.Methods: Two rhesus macaques and thirteen Dorset sheep were studied. FUS neuromodulation was targeted to the primary visual cortex in rhesus macaques and to subcortical locations, verified by MR-ARFI, in eleven sheep. Both rhesus macaques and five sheep received a single FUS session, whereas six sheep received repeated sessions three to six days apart. The remaining two control sheep did not receive ultrasound but otherwise underwent the same anesthetic and MRI procedures as the eleven experimental sheep.Hematoxylin and eosin-stained sections of brain tissue (harvested zero to eleven days fol- * Corresponding Author: lowing FUS) were evaluated for tissue damage at FUS and control locations as well as tissue within the path of the FUS beam. TUNEL staining was used to evaluate for the presence of apoptosis in sheep receiving high dose FUS.Results: No FUS-related pre-mortem histologic findings were observed in the rhesus macaques or in any of the examined sheep. Extravascular red blood cells (RBCs) were present within the meninges of all sheep, regardless of treatment group. Similarly, small aggregates of perivascular RBCs were rarely noted in non-target regions of neural parenchyma of FUS-treated (8/11) and untreated (2/2) sheep. However, no concurrent histologic abnormalities were observed, consistent with RBC extravasation occurring as post-mortem artifact following brain extraction. Sheep within the high dose FUS group were TUNELnegative at the targeted site of FUS.
Conclusions:The absence of FUS-related histologic findings suggests that the neuromodulation and MR-ARFI protocols evaluated do not cause tissue damage.We evaluate histology in brain tissue following FUS neuromodulation in the visual 49 cortex of rhesus macaques, and following neuromodulation and MR-ARFI in subcortical 50 brain regions in sheep. The sheep histology includes a treatment control group in which 51 4 no FUS was applied, and internal controls from hemispheres not treated with FUS. Our 52 neuromodulation protocols included a component similar to those used in human stud-53 ies, and to those evaluated by Lee and colleagues. We also investigated a broader range 54 of intensity values and repeated number of FUS bursts, exceeding those values typically 55 used in human protocols as well as those used in the study by Lee et al. Our findings 56 provide important information for subsequent studies involving FUS neuromodulation or 57 MR-ARFI. 58 Materials and Methods 59 All animal experiments were performed with institutional approval from the Stanford 60 University Administrative Panel on Laboratory Animal Care. 61 Rhesus macaque s...