Theranostics 2018; 8(8):2249-2250. doi:10.7150/thno.25468


Reply to Kovacs et al.: Concerning acute inflammatory response following focused ultrasound and microbubbles in the brain

Dallan McMahon1,2 Corresponding address, Kullervo Hynynen1,2,3

1. University of Toronto, Department of Medical Biophysics, Toronto, M4N 3M5, Canada;
2. Sunnybrook Research Institute, Toronto, M4N 3M5, Canada;
3. University of Toronto, Institute of Biomaterials and Biomedical Engineering, Toronto, M5S 3G9, Canada.

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license ( See for full terms and conditions.
McMahon D, Hynynen K. Reply to Kovacs et al.: Concerning acute inflammatory response following focused ultrasound and microbubbles in the brain. Theranostics 2018; 8(8):2249-2250. doi:10.7150/thno.25468. Available from

File import instruction

Letter in Theranostics, Volume 8, 2245


We thank Kovacs et al. for their editorial [15] on our recent publication [1] regarding the influence of microbubble (MB) dose on acute inflammatory response (AIR) following focused ultrasound (FUS)-induced increases in blood-brain barrier (BBB) permeability. We wish to address key points of disparity in interpretations of the data presented by us and in a previous report from Kovacs et al [2].

All data published to date indicate that an AIR follows increased BBB permeability induced by FUS + MBs [1-5]; however, there are discrepancies in the reported magnitude and duration of this response. To address this, we approximated sonication parameters shown by Kovacs et al. in PNAS to induce a substantial AIR [2] and compared this to parameters that better reflect those used in ongoing clinical trials ( identifiers: NCT02343991, NCT02986932, NCT03119961) and previous preclinical research [6-10]. Results indicated that MB dose and acoustic pressure impacts the degree of AIR, as measured by changes in the expression of NFκB pathway-related genes. We demonstrated a high degree of correlation between gene expression changes reported by Kovacs et al. and those measured in the sonication scheme designed to approximate this work (r2 = 0.84; p = 0.00001). Importantly, when MB dose was reduced, and peak negative pressure was calibrated to avoid inertial cavitation, a substantial reduction in the magnitude of AIR was observed. We believe this demonstrates that the permeability of the BBB can be transiently increased using FUS + MBs with a minimal AIR, as well as highlights the need for both careful attention to sonication parameters and the use of acoustic feedback control.

In their editorial, Kovacs et al. discuss differences in sonication parameters between the two studies [1,2] and conclude that these differences preclude a direct comparison of results. The first major difference is in MB dose. To approximate equivalent doses, necessitated by a disparity in MB type between the two studies, we used clinical imaging dose as a normalizing factor. Optison was administered at ~500 µL/kg versus Definity administered at 100 µL/kg, in Kovacs and McMahon, respectively. Both doses equate to 10 times the clinical imaging dose of their respective MB type. Kovacs et al. correctly highlight differences in MB number/kg between studies. However, using MB number as a method of comparing doses of different MB types necessarily considers all cavitation nuclei as equivalent; the assumption that each Definity MB will respond like an Optison MB, or vice versa, is somewhat over simplistic since differences in their shell properties and size alter their response to ultrasound [11,12]. Additionally, McDannold et al. have previously demonstrated that the probability of increased blood-brain barrier (BBB) permeability following FUS is approximately equivalent for Definity and Optison at their respective clinical imaging doses over a range of pressures [6].

Kovacs et al. also point out differences in anesthesia carrier gases and infusion rates between studies, both of which affect the number of MBs present in circulation while FUS is delivered. Using this as a metric to compare experimental conditions, they conclude that the number of MBs/kg present in circulation is lower in Kovacs et al. than in McMahon and Hynynen (scheme 2; designed to approximate conditions in Kovacs et al.). Again, this analysis considers every cavitation nuclei as equivalent; however, the smaller mean diameter of Definity MBs would contribute to a reduced impact on BBB permeability at this frequency relative to Optison MBs [12,13].

While it is important to note that there are several differences in experimental parameters, ultimately, the high degree of correlation in differential gene expression measured in the two studies suggests that the biological responses were very similar. We believe that the work presented in both Kovacs et al. [2] and McMahon and Hynynen [1] highlight the importance of optimizing sonication parameters for the desired effect with careful analysis of AIR especially for repeated treatments, as well as the necessity of using acoustic emissions to calibrate and control the applied ultrasound pressure. The continued development, refinement, and careful study of FUS + MBs for increasing BBB permeability is an important endeavor for advancing this technique into clinical implementation and for assessing the spectrum of its safety profile.


AIR: acute inflammatory response; BBB: blood-brain barrier; FUS: focused ultrasound; MB: microbubbles


The authors would like to extend their thanks to Dr. Ryan Jones and Charissa Poon for consultation. Funding for this work was provided by The National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (R01 EB003268), The Canadian Institutes for Health Research (FRN 119312), and the Canada Research Chair Program (awarded to KH).

Competing Interests

KH is the founder of FUS Instruments, from which he receives non-study related support. DM declares no competing financial interests.


1. McMahon D, Hynynen K. Acute inflammatory response following increased blood-brain barrier permeability induced by focused ultrasound is dependent on microbubble dose. Theranostics. 2017;7:3989-4000

2. Kovacs ZI, Kim S, Jikaria N, Qureshi F, Milo B, Lewis BK. et al. Disrupting the blood-brain barrier by focused ultrasound induces sterile inflammation. Proc Natl Acad Sci USA. 2017;114:E75-E84

3. McMahon D, Bendayan R, Hynynen K. Acute effects of focused ultrasound-induced increases in blood-brain barrier permeability on rat microvascular transcriptome. Sci Rep. 2017;7:45657

4. Jordão JF, Thévenot E, Markham-Coultes K, Scarcelli T, Weng Y-Q, Xhima K. et al. Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound. Exp Neurol. 2013;248:16-29

5. Leinenga G, Götz J. Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer's disease mouse model. Sci Transl Med. 2015;7:278ra33

6. McDannold N, Vykhodtseva N, Hynynen K. Use of ultrasound pulses combined with Definity for targeted blood-brain barrier disruption: a feasibility study. Ultrasound Med Biol. 2007;33:584-590

7. Park J, Zhang Y, Vykhodtseva N, Jolesz FA, McDannold NJ. The kinetics of blood brain barrier permeability and targeted doxorubicin delivery into brain induced by focused ultrasound. J Control Release. 2012;162:134-142

8. McDannold N, Arvanitis CD, Vykhodtseva N, Livingstone MS. Temporary disruption of the blood-brain barrier by use of ultrasound and microbubbles: safety and efficacy evaluation in rhesus macaques. Cancer Res. 2012;72:3652-3663

9. Burgess A, Dubey S, Yeung S, Hough O, Eterman N, Aubert I. et al. Alzheimer disease in a mouse model: MR imaging-guided focused ultrasound targeted to the hippocampus opens the blood-brain barrier and improves pathologic abnormalities and behavior. Radiology. 2014;273:736-745

10. O'Reilly MA, Hynynen K. Blood-brain barrier: real-time feedback-controlled focused ultrasound disruption by using an acoustic emissions-based controller. Radiology. 2012;263:96-106

11. van der Meer SM, Dollet B, Voormolen MM, Chin CT, Bouakaz A, de Jong N. et al. Microbubble spectroscopy of ultrasound contrast agents. J Acoust Soc Am. 2007;121:648-656

12. de Jong N, Hoff L, Skotland T, Bom N. Absorption and scatter of encapsulated gas filled microspheres: theoretical considerations and some measurements. Ultrasonics. 1992;30:95-103

13. Choi JJ, Feshitan JA, Baseri B, Wang S, Tung YS, Borden MA. et al. Microbubble-Size Dependence of Focused Ultrasound-Induced Blood-Brain Barrier Opening in Mice In Vivo. IEEE Transactions on Biomedical Engineering. 2010;57:145-154

14. Song K-H, Fan AC, Hinkle JJ, Newman J, Borden MA, Harvey BK. Microbubble gas volume: A unifying dose parameter in blood-brain barrier opening by focused ultrasound. Theranostics. 2017;7:144-152

15. Kovacs ZI, Burks SR, Frank JA. Focused ultrasound with microbubbles induces sterile inflammatory response proportional to the blood brain barrier opening: Attention to experimental conditions. Theranostics. 2018;8(8):2245-2248

Author contact

Corresponding address Corresponding author:

Received 2018-2-9
Accepted 2018-2-16
Published 2018-3-8