Table 1.

Sample of safety studies for FUS-mediated BBBO. This table summarizes the FUS parameters, experimental designs, and safety results from a selection of papers. Papers that used similar FUS parameters and experimental designs are grouped together. The asterisk (*) indicates that FUS was applied transcranially; otherwise, animals underwent a craniotomy prior to FUS treatment.

Species	Frequency	Peak acoustic pressure	MB brand (dose)	Time of sacrifice	Evaluation method	Safety results	Reference
Rabbit	1.63 MHz	Up to ~ 4.6 MPa	Optison (50 μL/kg)	2 h–4 wks after sonication	Contrast-enhanced MRI Light microscopy: H&E, vanadium acid fuchsin-toluidine blue, TUNEL	0.7 – 1 MPa: BBBO, but no neuronal damage 2–3 MPa: 25% of locations had neuronal damage >4 MPa: 70% of locations had neuronal damage Few extravasated RBCs, few apoptotic or ischemic cells around sonicated locations No delayed effects observed by MRI or histology 4 wks after treatment	Hynynen et al., 2001 – Noninvasive MRI-guided focal opening of the BBB in rabbits McDannold et al., 2005 – MRI-guided targeted BBB disruption with FUS: Histological findings in rabbits
Rabbit	0.69 MHz	Up to 3.1 MPa	Optison (50–250 μL/kg) Definity (10 μL/kg, used as well in McDannold et al., 2007)	4–48 h after sonication	Contrast-enhanced MRI Light microscopy: H&E, TUNEL, vanadium acid fuchsin-toluidine blue Electron microscopy	MRI contrast enhancement increased with acoustic pressure, BBBO evident starting at 0.4 MPa Brain tissue necrosis at 70–80% of sonicated locations if ≥ 2.3 MPa Optison produced greater effect than Definity at same acoustic pressure amplitudes (McDannold et al., 2006) Magnitude of BBBO increased with higher burst length, but was not significantly affected by PRF or Optison dosages tested	Hynynen et al., 2005 – Local and reversible blood-brain barrier disruption using FUS at frequencies suitable for trans-skull sonications McDannold et al., 2007 – Use of US pulses combined with Definity for targeted BBB disruption – a feasibility study McDannold et al., 2008b – Effect of acoustic parameters and ultrasound contrast agent dose on FUS induced BBB disruption
Rabbit	2.04 MHz	0.3–2.3 MPa	Optison (50 μL/kg)	4 h after last sonication	Contrast-enhanced MRI Light microscopy: H&E	Few to no RBC extravasation at 2.04 MHz Density of RBC extravasations significantly increased at higher frequency (1.63 MHz) than at lower frequencies (representative H&E images taken from other experiments) MI threshold for BBBO = 0.46	McDannold et al., 2008b – BBB disruption induced by FUS and circulating preformed MBs appears to be characterized by the MI
Rabbit	0.26 MHz	0.11–0.57 MPa	Optison (50 μL/kg)	4 h after sonication	Contrast-enhanced MRI Light microscopy: H&E	BBBO observed in contrast MRI at 0.29–0.57 MPa, associated with few to no RBC extravasation	McDannold et al., 2006 – Targeted disruption of the BBB with FUS – association with cavitation activity
Rat	1 MHz	N/A	SonoVue (150–450 μL/kg)	Up to 4 h after sonication	Contrast-enhanced MRI Evans blue	Greater MB doses led to greater magnitude of BBBO	*Yang et al., 2009 – Effect of ultrasound contrast agent dose on the duration of focused-ultrasound-induced BBB disruption
Rat	0.558 MHz	0.3 MPa	Definity (20 μL/kg)	Immediately after experiment	Contrast-enhanced MRI Immunoblot Fluorescence microscopy: p-Akt, p-GSK3β, GFAP (astrocytes), NeuN (neurons), ZO-1 (TJ protein), von Willebrand Factor (endothelial damage)	Extravasated IgG in sonicated regions Decreased interaction of occluden and ZO-1 (TJ proteins) but no loss of occluden or ZO-1 levels, increased PI3K/Akt signaling, no change in MAPK signaling, in sonicated regions Increased p-Akt and p-GSK3β levels in neurons around opened blood vessels	*Jalali et al., 2010 – FUS-mediated BBB disruption is associated with increase in Akt activation in rats
Rat	0.5515 MHz	Acoustic pressure increased incrementally until ultraharmonic emissions detected	Definity (20 μL/kg)	2 h–8 d after sonication	Contrast-enhanced MRI Light microscopy: H&E Immunohistochemistry: NeuN (neurons)	0.28 MPa ± 0.05 sonications resulted in BBBO Scaling pressures by 50% after ultraharmonics detected allowed BBBO without causing edema, some to no RBC extravasation, no vacuolations No neuronal loss 8 d after sonication	*O'Reilly and Hynynen 2012 – BBB real-time feedback-controlled FUS disruption by using an acoustic emissions based controller
Rat	1.5 MHz	0.45 MPa	Sonovue (1.5 × 108 MB/mL, 200 μL)	N/A	Contrast-enhanced MRI	BBB closed at progressively slower rate, hypothesized to be due to transcellular passage between ECs	*Marty et al., 2012 – Dynamic study of BBB closure after its disruption using ultrasound: a quantitative analysis
Rat	0.4 MHz	0.2–0.5 MPa	SonoVue (100 μL/kg)	1 h–1 wk after sonication	Somatosensory evoked potentials (SSEPs) Functional MRI for blood-oxygen-level dependent (BOLD) measurements Light microscopy: H&E Immunohistochemistry: NeuN (neurons), TuJ1 (anti-neuron specific class III ß-tubulin) Evans blue	No BBBO at 0.2 MPa, but BBBO at 0.35 MPa and 0.5 MPa sonication RBC extravasation highest in 0.5 MPa treated animals FUS at 0.5 MPa suppressed SSEP amplitude and prolonged latency for as long as 1 wk; at 0.35 MPa, SSEP suppression was observed for < 1h	*Chu et al., 2015 – Neuromodulation accompanying FUS-induced BBB opening
Rat	0.69 MHz	0.66–0.80 MPa	Definity (10 μL/kg)	1–36 h after last sonication	Contrast-enhanced MRI Light microscopy: H&E, Von Kossa (mineralization), GFAP (astrocytes)	Contrast-enhanced T2-weighted images, but not T1-weighted images, corresponded well with histology results 0.73 MPa: Few to no microhemorrhages observed after six sonications 0.80 MPa: Microhemorrhages and some acute ischemic and necrotic damage observed after six sonications Micro-scars consisting of reactive astrocytes observed in some animals	*Kobus et al., 2016 – Safety validation of repeated BBB disruption using FUS
Mouse	1.525 MHz	0.15–0.98 MPa	Definity (50 μL/kg)	Within 5 h of sonication	Contrast-enhanced MRI Fluorescence microscopy: Tracer injection (3 kDa Texas Red dextran) Light microscopy: H&E	Fluorescence microscopy and MRI of tracers: BBBO threshold between 0.15 and 0.30 MPa Safest BBBO between 0.3 and 0.46 MPa 0.3 MPa: few RBC extravasation, few small microvacuolated areas 0.46 MPa: more RBC extravasations, presence of dark neurons and microvacuolations Damage increased with increasing acoustic pressure	*Baseri et al., 2010 – Multi-modality safety assessment of BBBO using FUS and Definity MBs: A short-term study
Mouse	1.525 MHz	0.30–0.60 MPa	In-house MBs: 1–2, 4–5, or 6–7 μm(1 μL/g × 107 MB/mL)	7 d after sonication	Contrast-enhanced MRI Light microscopy: H&E	Magnitude of BBBO increased with sonication pressure and MB diameter BBB closure time proportional to magnitude of BBBO (24 h −5 d after sonication) Cell loss correlated with hypointensity in MRI	*Samiotaki et al., 2012 – A quantitative pressure and MB size dependence study of FUS-induced BBBO reversibility in vivo using MRI
Mouse	1 MHz	0.7 MPa	In-house MBs (1–5×107 MBs/mL, injected 1 μL/g retroorbitally)	30 min–1 h after sonication	Light microscopy: H&E, Nissl, vanadium acid fuchsin Evans blue Fluorescence microscopy: GFAP (astrocytes), IBA1 (microglia)	No degenerating neurons, edema, or RBC extravasation No ischemic damage Microglial activation, but no astrogliosis, at one an 24 h after sonication	*Leinenga and Gotz 2015 – Scanning ultrasound removes Aß and restores memory in an AD mouse model
Rhesus macaque	0.5 MHz	0.3–0.6 MPa	Definity (1.2 × 1010 MBs/mL) and in-house MBs (5 × 109 MB/mL) 500 μL MBs injected per animal	N/A	Contrast-enhanced MRI	0.3 MPa sufficient to cause BBBO	*Marquet et al., 2011 – Noninvasive, transient and selective BBBO in non-human primates in vivo
Rhesus macaque	ExAblate 4000 (InSightec, 2015) phased array 0.22 MHz	N/A	Definity (10–20 μL/kg)	2 h–2 wks after last sonication	Contrast-enhanced MRI Functional tests Light microscopy: Nissl, H&E, Luxol Fast Blue, Bielschowsky's silver stain, Prussian blue, TUNEL Trypan blue	BBBO varied depending on area of brain targeted No functional impairments after single or repeated BBBO in visual areas over several weeks BBBO probability 50% at 149 kPa Tissue damage probability 50% at 300 kPa	*McDannold et al., 2012 – Temporary disruption of the BBB by use of ultrasound and MBs: Safety and efficacy evaluation in rhesus monkeys

^*transcranial US.