Table 1.

Summary of experiments investigating the biophysical impacts of therapeutic ultrasound, where all effects are compared to a non-US group (i.e. LIPUS compared to SHAM, LPS+LIPUS compared to SHAM+LIPUS). For a more extensive compilation of the effect of LIPUS across cell types, see the review by Xu et al. ⁷.

Model	Experiment	LIPUS Parameters	Biological effect
in vitro 76 macrophages: bone marrow derived HUS	measured gene expression 3h post-US LPS: 100ng/mL measured inflammatory response to LPS 3h post-US, 1.5h with LPS (4.5h from start of US)	fc: 1 MHz fR: 100 Hz ISPTA: 3 W/cm2 Duration: 5 min Duty cycle: 20% Burst length: N/A Transducer: plane	US: - apoptosis, ↑Irg1 (mRNA), ↑itaconate (mRNA), ↑Nrf2 (mRNA) US+LPS: ↓IL-1b (mRNA), ↓IL-6 (mRNA), ↓Tnfα (mRNA) Verified cell media did not exceed 37 oC
in vitro 81 microglia: BV-2 astrocytes: CTX TNA2 LIPUS	LPS: 1 μg/mL LIPUS 12h post-LPS Evaluation at 4, 8, 12, 24 h post-LIPUS	fc: 1 MHz fR: 100 Hz ISPTA: 10, 20, 30 mW/cm2 Duration: 15 min (3x5/5 on/off) Duty cycle: 50% Burst length: 5 ms Transducer: plane	Microglia LIPUS: ↑BDNF, ↑GDNF, ↑VEGF, ↑p-CREB/CREB, ↑p-Akt/Akt, ↑p-TrkB/TrkB LPS+LIPUS: ↑BDNF, ↑GDNF, ↓VEGF, ↓TNFa, ↓IL-1b, ↓IL-6, ↑p-CREB/CREB, ↑p-Akt/Akt, ↑p-TrkB/TrkB, ↓TLR4, ↓MyD88, ↓p-JNK/JNK, ↓p-p65/p65, ↓iNOS TrkB/Akt/CREB/BDNF pathway required for BDNF production Akt inhibits JNK Astrocytes LIPUS: ↑BDNF, ↑GDNF, ↑VEGF,
in vitro 84 astrocytes: CTX TNA2 LIPUS	Evaluation at 4, 8, 12, 24 h post-LIPUS	fc: 1 MHz fR: 10 Hz ISPTA: 110 mW/cm2 Duration: 15 min (3x5/5 on/off) Duty cycle: 50% Burst length: 50 ms Transducer: plane	LIPUS: ↑BDNF, ↑p-TrkB/TrkB, ↑p-Akt/Akt, ↑Ca2+ influx, ↓p-CREB/CREB, ↑p-NF-kB p65/NF-kB p65, ↑p-p65/p65
in vitro 76 preosteoblasts: MC3T3-E1 LIPUS	mRNA measurement 0, 6, 24, 48h post-LIPUS	fc: 1.5 MHz fR: 1 kHz ISATA: 30 mW/cm2 Duration: 20 min Duty cycle: 25% Burst length: 0.2 s Transducer: N/A	LIPUS: ↑CD200 (mRNA) at 24h, ↑bone morphology genes
in vitro 190 osteoblasts: MC3T3-E1 LIPUS	LPS: 1 μg/mL LIPUS/sham simultaneous with LPS	fc: 1.5 MHz fR: 100 kHz ISATA: 30 mW/cm2 Duration: 2 h Duty cycle: N/A Burst length: 0.2 ms Transducer: N/A	LPS: ↑CCL2 (mRNA), ↑CXCL1 (mRNA), ↑ CXCL10 (mRNA), ↑RANKL (mRNA), ↑NF-kB (mRNA) LPS+LIPUS: - CCL2 (mRNA), ↓CXCL1 (mRNA), ↓CXCL10 (mRNA), ↓RANKL (mRNA), ↓NF-kB (mRNA), ↓ERK/Akt/IKK/IRF3, ↓TLR4/MyD88 association
in vitro 114 endothelial: ECV 304	Applied laminar fluid shear stress to endothelial cell line	Laminar fluid shear stress: 15 dynes/cm2 (1.6 Pa) Duration: 5, 15, 30, 60 mins	Shear Stress: prolonged shear stress led to increased association between eNOS and HSP90, but did not lead to an increase in HSP90
in vivo 77 Female Sprague-Dawley rats LIPUS	6-OHDA: 12μg 2μL of 6-OHDA injected into right striatum 6-OHDA model of Parkinson's Disease Cellular evaluation at 8 weeks post-injection	fc: 1 MHz fR: 1 Hz ISPTA: 528 mW/cm2 Duration: 15 min (3x5/5 on/off) Duty cycle: 5% Burst length: 50 ms Transducer: focused, single hemisphere	6-OHDA+LIPUS: ↓GFAP+ astrocytes, ↓Iba1+ microglia, ↓IL-1b, ↓p-NF-kB, ↑GDNF, - BDNF (insignificant increase), ↑BBB stability (ZO-1, claudin-5) LIPUS started 2 weeks following 6-OHDA injection, LIPUS administered 5 days/week for next 6 weeks Observed no tissue damage with LIPUS regimen
in vivo 97 Beagles (2-8 years old) LIPUS	Wound healing in mucoperiosteal flap surgery	fc: 1.5 MHz fR: 1 kHz I: 30 mW/cm2 Duration: 20 min/day Duty cycle: N/A Burst length: 0.2 ms Transducer: 13 mm diameter	LIPUS: ↑HSP70 expression in gingival tissues (absent in control), ↑cementum and mandibular bone regeneration LIPUS continued for period of 4 weeks following surgery
in vivo 78 Female C57BL/6 mice (7 weeks old) LIPUS	LIPUS in model of repetitive restraint stress (RRS): 22 days LIPUS in cuprizone (0.2% in chow) model of demyelination: 35 days Analysis 24 hours following last LIPUS treatment	fc: 1.5 MHz fR: 1 kHz ISPTA: 25 mW/cm2 Duration: 20 min/day Duty cycle: 20% Burst length: N/A Transducer: plane	LIPUS: ↑BDNF (hippocampus) RRS+LIPUS: ↓anhedonia, ↑BDNF (hippocampus) CUPRIZONE+LIPUS: ↑MBP (myelin basic protein, cortex), ↑NG2 (neural/glial antigen 2, cortex) NG2 expressed in oligodendrocyte progenitors, and microglia
in vivo 79 Male C57BL/6 mice (8 weeks old) LIPUS	LPS: 250 μg/kg LPS administration from days 5-11 LIPUS treatment from days 7-13 Biological analysis at 4 (GFAP, Iba1, BDNF, CREB) and 48 h post-LIPUS	fc: 1 MHz fR: 1 Hz ISPTA: 528 mW/cm2 Duration: 15 min (3x5/5 on/off) Duty cycle: 5% Burst length: N/A Transducer: focal, (D: 38 mm, ROC:63.5 mm; half-max pressure amplitude of focal zone - D: 3 mm, L: 26 mm)	LPS+LIPUS: ↑spatial learning and memory (Morris water maze, novel object recognition), ↓Aβ (hippocampus, but not cortex), ↓cleaved caspase-3, ↓GFAP, - Iba1 (non-significant decrease), ↓TNFa, ↓IL-1b, ↓IL-6, ↓p-NF-kB, ↓TLR4, ↑BDNF, ↑p-CREB LIPUS: ↑BDNF, ↑p-CREB, ↑p-NF-kB (cortex), ↑TLR4 Increase in TLR4 and p-NF-kB in LIPUS group did not correspond to a significant increase in TNFa, IL-1b, or IL-6
in vivo 64 Vascular Dementia Male C57BL/6 mice (10-12 weeks old), eNOS-/- (C57BL/6 background) Alzheimer's Disease Male 5XFAD transgenic mice (14-16 weeks old, C57BL/6 background) LIPUS	LIPUS applied every other day following surgery (i.e. 1, 3, 5; BCAS or sham for vascular dementia) LIPUS applied on days: 1, 3, 5, 28, 30, 32, 56, 58, 60, 84, 86 for AD model, testing on day 86	fc: 1.875 MHz fR: 6 kHz ISPTA: 90 mW/cm2 Duration: 20 min (x3: right, midline, left)/day Duty cycle: 5% Burst length: 17μs Transducer: focal, (focus: 9 cm, beam width: 3.6-4 mm)	VaD+LIPUS - Molecular (day 3/7): ↓WM lesions, ↑angiogenesis and OPC genes (mRNA), ↑eNOS (both inactivated and activated; correlated with increased NGF, BDNF, VEGF), ↑GFAP, ↑Olig2+ VaD+LIPUS - Behavioural (day 28): ↑cognitive function (Y-maze, passive avoidance, novel object recognition, not all significant) VaD+LIPUS - CBF: ↑CBF with LIPUS over time (start at day 2, continues to day 28) VaD+LIPUS+eNOS-/- (with respect to VaD): - behavioural, - CBF, - WM lesions, - NGF, - BDNF, - VEGF 5XFAD+LIPUS: ↑HSP90, ↑CBF, ↑immune and angiogenesis genes (mRNA), ↑cognitive function (Y-maze), ↓Iba1+, ↑eNOS, ↑BDNF, ↓Aβ Note: HSP90 not measured in VaD group, only 5XFAD
in vivo 80 Male C57BL/6 mice (8 weeks old) LIPUS	Cortical Impact Injury (CCI) - model of traumatic brain injury (TBI) LIPUS applied 5 mins post-CCI, daily thereafter Analysis at day 1, 4, and 148	fc: 1 MHz fR: N/A ISPTA: 528 mW/cm2 Duration: 5 min/day Duty cycle: N/A Burst length: N/A Transducer: focal, (focal diameter: 3 mm, focal width: 26 mm, diameter: 38 mm, ROC: 63.5 mm)	TBI+LIPUS (day 1): -MMP-9, ↓neutrophils, ↓Iba1+, -p-Foxo1, ↓neurological deficits TBI+LIPUS (day 4): ↓MMP-9, ↓neutrophils, ↓Iba1+, ↑p-Foxo1, ↓neurological deficits TBI+LIPUS (day 148): insignificant ↑BDNF+ neurons, ↑neurons, ↓T2w edema volume, ↓neurological deficits