Abstract
Focused ultrasound (FUS) with microbubbles opens the blood-brain barrier (BBB) for targeted drug delivery into the brain. How brain endothelial cells (BECs) respond to low acoustic pressures known to open the BBB transiently, or high pressures that cause brain damage, is incompletely characterized. Here, we apply FUS at low (450 kPa) and high (750 kPa) pressures in mice where BBB tight junctions are labeled with eGFP to characterize their abnormalities. Arteriole and capillary BECs respond to low pressure by a transient BBB tight junction reorganization and opening. In contrast, high pressure induces tight junction obliteration and persistent BBB opening in BECs even after 72 hours, associated with microglial activation. Transcriptomic analyses of BECs show that high pressure upregulates genes related to the stress response and cell junction disassembly, whereas low pressure upregulates intracellular repair genes. Thus, transient reorganization and repair of tight junctions mediate safe BBB opening for therapeutic delivery.
Similar content being viewed by others
Data availability
The raw and analyzed mouse BEC scRNA-seq datasets supporting this manuscript are archived at the NIH GEO repository under accession number GSE253425. The source data files for the Main and Extended Data figures associated with this manuscript can be provided to the readers upon request.
Code availability
No commercial code was used in the manuscript. We can provide the code used in R for the analysis of single cell RNA sequencing data to the readers upon request.
References
Ben-Zvi, A. et al. Mfsd2a is critical for the formation and function of the blood-brain barrier. Nature 509, 507–511 (2014).
Andreone, B. J. et al. Blood-brain barrier permeability is regulated by lipid transport-dependent suppression of caveolae-mediated transcytosis. Neuron 94, 581–594.e5 (2017).
Lutz, S. E. et al. Caveolin1 is required for Th1 cell infiltration, but not tight junction remodeling, at the blood-brain barrier in autoimmune neuroinflammation. Cell Rep. 21, 2104–2117 (2017).
Liebner, S. et al. Functional morphology of the blood-brain barrier in health and disease. Acta Neuropathol. (Berl.) 135, 311–336 (2018).
Biswas, S., Cottarelli, A. & Agalliu, D. Neuronal and glial regulation of CNS angiogenesis and barriergenesis. Dev. Camb. Engl. 147, dev182279 (2020).
Meng, Y. et al. Safety and efficacy of focused ultrasound induced blood-brain barrier opening, an integrative review of animal and human studies. J. Controlled Release 309, 25–36 (2019).
Leighton, T. G. The Acoustic Bubble (Academic Press, 1997).
Miller, D. L. Particle gathering and microstreaming near ultrasonically activated gas-filled micropores. Cit. J. Acoust. Soc. Am. 84, 1378 (1988).
Kodama, T. & Tomita, Y. Cavitation bubble behavior and bubble-shock wave interaction near a gelatin surface as a study of in vivo bubble dynamics. Appl. Phys. B Lasers Opt. 70, 139–149 (2000).
Prentice, P., Cuschieri, A., Dholakia, K., Prausnitz, M. & Campbell, P. Membrane disruption by optically controlled microbubble cavitation. Nat. Phys. 1, 107–110 (2005).
Qin, S. & Ferrara, K. W. Acoustic response of compliable microvessels containing ultrasound contrast agents. Phys. Med. Biol. 51, 5065–5088 (2006).
McDannold, N., Vykhodtseva, N. & Hynynen, K. Blood-brain barrier disruption induced by focused ultrasound and circulating preformed microbubbles appears to be characterized by the mechanical index. Ultrasound Med. Biol. 34, 834–840 (2008).
Vlachos, F., Tung, Y.-S. & Konofagou, E. Permeability dependence study of the focused ultrasound-induced blood-brain barrier opening at distinct pressures and microbubble diameters using DCE-MRI. Magn. Reson. Med. 66, 821–830 (2011).
Samiotaki, G., Vlachos, F., Tung, Y.-S. & Konofagou, E. E. A quantitative pressure and microbubble-size dependence study of focused ultrasound-induced blood-brain barrier opening reversibility in vivo using MRI. Magn. Reson. Med. 67, 769–777 (2012).
O’Reilly, M. A., Hough, O. & Hynynen, K. Blood-brain barrier closure time after controlled ultrasound-induced opening is independent of opening volume. J. Ultrasound Med. J. Am. Inst. Ultrasound Med. 36, 475–483 (2017).
Samiotaki, G. & Konofagou, E. E. Dependence of the reversibility of focused- ultrasound-induced blood-brain barrier opening on pressure and pulse length in vivo. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 60, 2257–2265 (2013).
O’Reilly, M. A., Waspe, A. C., Ganguly, M. & Hynynen, K. Focused-ultrasound disruption of the blood-brain barrier using closely-timed short pulses: influence of sonication parameters and injection rate. Ultrasound Med. Biol. 37, 587–594 (2011).
Choi, J. J. et al. Noninvasive and localized blood-brain barrier disruption using focused ultrasound can be achieved at short pulse lengths and low pulse repetition frequencies. J. Cereb. Blood Flow. Metab. J. Int. Soc. Cereb. Blood Flow. Metab. 31, 725–737 (2011).
Choi, J. J. et al. Microbubble-size dependence of focused ultrasound-induced blood–brain barrier opening in mice In Vivo. IEEE Trans. Biomed. Eng. 57, 145–154 (2010).
McMahon, D. & Hynynen, K. Acute inflammatory response following increased blood-brain barrier permeability induced by focused ultrasound is dependent on microbubble dose. Theranostics 7, 3989–4000 (2017).
Marquet, F., Tung, Y.-S., Teichert, T., Ferrera, V. P. & Konofagou, E. E. Noninvasive, transient and selective blood-brain barrier opening in non-human primates in vivo. PLoS One 6, e22598 (2011).
McDannold, N., Arvanitis, C. D., Vykhodtseva, N. & Livingstone, M. S. Temporary disruption of the blood-brain barrier by use of ultrasound and microbubbles: safety and efficacy evaluation in rhesus macaques. Cancer Res. 72, 3652 (2012).
Mainprize, T. et al. Blood-brain barrier opening in primary brain tumors with non-invasive MR-guided focused ultrasound: a clinical safety and feasibility study. Sci. Rep. 9, 321 (2019).
Lipsman, N. et al. Blood–brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nat. Commun. 9, 2336 (2018).
Treat, L. H. et al. Targeted delivery of doxorubicin to the rat brain at therapeutic levels using MRI-guided focused ultrasound. Int. J. Cancer 121, 901–907 (2007).
Park, E.-J., Zhang, Y.-Z., Vykhodtseva, N. & McDannold, N. Ultrasound-mediated blood-brain/blood-tumor barrier disruption improves outcomes with trastuzumab in a breast cancer brain metastasis model. J. Controlled Release 163, 277–284 (2012).
Kinoshita, M., McDannold, N., Jolesz, F. A. & Hynynen, K. Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption. Proc. Natl. Acad. Sci. USA 103, 11719–11723 (2006).
Jordão, J. F. et al. Antibodies targeted to the brain with image-guided focused ultrasound reduces amyloid-β plaque load in the TgCRND8 mouse model of Alzheimer’s disease. PLoS ONE 5, e10549 (2010).
Kinoshita, M., McDannold, N., Jolesz, F. A. & Hynynen, K. Targeted delivery of antibodies through the blood–brain barrier by MRI-guided focused ultrasound. Biochem. Biophys. Res. Commun. 340, 1085–1090 (2006).
Samiotaki, G., Acosta, C., Wang, S. & Konofagou, E. E. Enhanced delivery and bioactivity of the neurturin neurotrophic factor through focused ultrasound—mediated blood—brain barrier opening in vivo. J. Cereb. Blood Flow. Metab. 35, 611–622 (2015).
Baseri, B. et al. Activation of signaling pathways following localized delivery of systemically administered neurotrophic factors across the blood–brain barrier using focused ultrasound and microbubbles. Phys. Med. Biol. 57, N65–N81 (2012).
Wang, S., Olumolade, O. O., Sun, T., Samiotaki, G. & Konofagou, E. E. Noninvasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus. Gene Ther. 22, 104–110 (2015).
Thévenot, E. et al. Targeted delivery of self-complementary adeno-associated virus serotype 9 to the brain, using magnetic resonance imaging-guided focused ultrasound. Hum. Gene Ther. 23, 1144–1155 (2012).
Burgess, A. et al. Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier. PloS One 6, e27877 (2011).
Sheikov, N., McDannold, N., Vykhodtseva, N., Jolesz, F. & Hynynen, K. Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med. Biol. 30, 979–989 (2004).
Sheikov, N., McDannold, N., Sharma, S. & Hynynen, K. Effect of focused ultrasound applied with an ultrasound contrast agent on the tight junctional integrity of the brain microvascular endothelium. Ultrasound Med. Biol. 34, 1093–1104 (2008).
Raymond, S. B., Skoch, J., Hynynen, K. & Bacskai, B. J. Multiphoton imaging of ultrasound/optison mediated cerebrovascular effects in vivo. J. Cereb. Blood Flow. Metab. 27, 393–403 (2007).
Nhan, T. et al. Drug delivery to the brain by focused ultrasound induced blood-brain barrier disruption: quantitative evaluation of enhanced permeability of cerebral vasculature using two-photon microscopy. J. Control. Release J. Control. Release Soc. 172, 274–280 (2013).
Knowland, D. et al. Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke. Neuron 82, 603–617 (2014).
Sun, T. et al. Acoustic cavitation-based monitoring of the reversibility and permeability of ultrasound-induced blood-brain barrier opening. Phys. Med. Biol. 60, 9079–9094 (2015).
Tung, Y.-S., Marquet, F., Teichert, T., Ferrera, V. & Konofagou, E. E. Feasibility of noninvasive cavitation-guided blood-brain barrier opening using focused ultrasound and microbubbles in nonhuman primates. Appl. Phys. Lett. 98, 163704 (2011).
Wang, S., Samiotaki, G., Olumolade, O., Feshitan, J. A. & Konofagou, E. E. Microbubble type and distribution dependence of focused ultrasound-induced blood-brain barrier opening. Ultrasound Med. Biol. 40, 130–137 (2014).
Choi, J. J. et al. Noninvasive and transient blood-brain barrier opening in the hippocampus of Alzheimer’s double transgenic mice using focused ultrasound. Ultrason. Imaging 30, 189–200 (2008).
Shahriar, S. et al. VEGF-A-mediated venous endothelial cell proliferation results in neoangiogenesis during neuroinflammation. Nat. Neurosci. 27, 1904–1917 (2024).
Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587.e29 (2021).
Menon, V. Clustering single cells: a review of approaches on high-and low-depth single-cell RNA-seq data. Brief. Funct. Genom. 17, 240–245 (2018).
Cembrowski, M. S. & Menon, V. Continuous variation within cell types of the nervous system. Trends Neurosci. 41, 337–348 (2018).
Hawrylycz, M. et al. Canonical genetic signatures of the adult human brain. Nat. Neurosci. 18, 1832–1844 (2015).
Sorensen, S. A. et al. Correlated gene expression and target specificity demonstrate excitatory projection neuron diversity. Cereb. Cortex N.Y. 25, 433–449 (2015).
Tasic, B. et al. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016).
Lengfeld, J. E. et al. Endothelial Wnt/β-catenin signaling reduces immune cell infiltration in multiple sclerosis. Proc. Natl. Acad. Sci. USA 114, E1168–E1177 (2017).
Platt, M. P. et al. Th17 lymphocytes drive vascular and neuronal deficits in a mouse model of postinfectious autoimmune encephalitis. Proc. Natl. Acad. Sci. USA 117, 6708–6716 (2020).
Karakatsani, M. E. et al. Amelioration of the nigrostriatal pathway facilitated by ultrasound-mediated neurotrophic delivery in early Parkinson’s disease. J. Controlled Release 303, 289–301 (2019).
Prosperetti, A. A general derivation of the subharmonic threshold for non-linear bubble oscillations. J. Acoust. Soc. Am. 133, 3719–3726 (2013).
Prakash, R. & Carmichael, S. T. Blood-brain barrier breakdown and neovascularization processes after stroke and traumatic brain injury. Curr. Opin. Neurol. 28, 556–564 (2015).
Protzmann, J., Jung, F., Jakobsson, L. & Fredriksson, L. Analysis of ischemic stroke-mediated effects on blood-brain barrier properties along the arteriovenous axis assessed by intravital two-photon imaging. Fluids Barriers CNS 21, 35 (2024).
Davalos, D. & Akassoglou, K. Fibrinogen as a key regulator of inflammation in disease. Semin. Immunopathol. 34, 43–62 (2012).
Davalos, D. et al. Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation. Nat. Commun. 3, 1227 (2012).
Adams, R. A. et al. The fibrin-derived γ377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease. J. Exp. Med. 204, 571–582 (2007).
Cortes-Canteli, M. et al. Fibrinogen and β-amyloid association alters thrombosis and fibrinolysis: a possible contributing factor to Alzheimer’s disease. Neuron 66, 695–709 (2010).
Petersen, M. A., Ryu, J. K. & Akassoglou, K. Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics. Nat. Rev. Neurosci. 19, 283–301 (2018).
Ryu, J. K. et al. Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation. Nat. Commun. 6, 8164 (2015).
Vanlandewijck, M. et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475–480 (2018).
Miao, Z. et al. Putative cell type discovery from single-cell gene expression data. Nat. Methods 17, 621–628 (2020).
Zarkada, G. et al. Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation. Dev. Cell 56, 2237–2251.e6 (2021).
Zhao, Q. et al. Single-cell transcriptome analyses reveal endothelial cell heterogeneity in tumors and changes following antiangiogenic treatment. Cancer Res 78, 2370–2382 (2018).
Caskey, C. F., Stieger, S. M., Qin, S., Dayton, P. A. & Ferrara, K. W. Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall. J. Acoust. Soc. Am. 122, 1191–1200 (2007).
Caskey, C. F., Kruse, D. E., Dayton, P. A., Kitano, T. K. & Ferrara, K. W. Microbubble oscillation in tubes with diameters of 12, 25, and 195 microns. Appl. Phys. Lett. 88, 1–3 (2006).
Zheng, H. et al. Ultrasound-driven microbubble oscillation and translation within small phantom vessels. Ultrasound Med. Biol. 33, 1978–1987 (2007).
Sassaroli, E. & Hynynen, K. Cavitation threshold of microbubbles in gel tunnels by focused ultrasound. Ultrasound Med. Biol. 33, 1651–1660 (2007).
Sassaroli, E. & Hynynen, K. Resonance frequency of microbubbles in small blood vessels: a numerical study. Phys. Med. Biol. 50, 5292–5305 (2005).
Qin, S. & Ferrara, K. W. The natural frequency of nonlinear oscillation of ultrasound contrast agents in microvessels. Ultrasound Med. Biol. 33, 1140–1148 (2007).
Sheikov, N. et al. Brain arterioles show more active vesicular transport of blood-borne tracer molecules than capillaries and venules after focused ultrasound-evoked opening of the blood-brain barrier. Ultrasound Med. Biol. 32, 1399–1409 (2006).
Shen, L., Weber, C. R. & Turner, J. R. The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state. J. Cell Biol. 181, 683–695 (2008).
Marchiando, A. M. et al. Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo. J. Cell Biol. 189, 111–126 (2010).
Beutel, O., Maraspini, R., Pombo-García, K., Martin-Lemaitre, C. & Honigmann, A. Phase separation of zonula occludens proteins drives formation of tight junctions. Cell 179, 923–936.e11 (2019).
Hynynen, K., McDannold, N., Sheikov, N. A., Jolesz, F. A. & Vykhodtseva, N. Local and reversible blood–brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications. NeuroImage 24, 12–20 (2005).
Deng, J. et al. The role of caveolin-1 in blood–brain barrier disruption induced by focused ultrasound combined with microbubbles. J. Mol. Neurosci. 46, 677–687 (2012).
Pandit, R. et al. Role for caveolin-mediated transcytosis in facilitating transport of large cargoes into the brain via ultrasound. J. Controlled Release 327, 667–675 (2020).
Wu, C.-C. et al. Blood-brain barrier opening with neuronavigation-guided focused ultrasound in pediatric patients with diffuse midline glioma Editor’s summary. Sci. Transl. Med. 17, https://doi.org/10.1126/scitranslmed.adq6645 (2025).
Bae, S. et al. Transcranial blood-brain barrier opening in Alzheimer's disease patients using a portable focused ultrasound system with real-time 2-D cavitation mapping. Theranostics 14, 4519–4535 (2024).
O’Reilly, M. A., Huang, Y. & Hynynen, K. The impact of standing wave effects on transcranial focused ultrasound disruption of the blood-brain barrier in a rat model. Phys. Med. Biol. 55, 5251–5267 (2010).
Tang, S. C. & Clement, G. T. Acoustic standing wave suppression using randomized phase-shift-keying excitations. J. Acoust. Soc. Am. 126, 1667 (2009).
Connor, C. W. & Hynynen, K. Patterns of thermal deposition in the skull during transcranial focused ultrasound surgery. IEEE Trans. Biomed. Eng. 51, 1693–1706 (2004).
Asquier, N. et al. Blood-brain barrier disruption in humans using an implantable ultrasound device: quantification with MR images and correlation with local acoustic pressure. J. Neurosurg. 1–9 https://doi.org/10.3171/2018.9.JNS182001 (2019).
Acknowledgements
We thank Shutao Wang and Camilo Acosta for their supporting role in this work. T.K., R.L.N., R.J. and E.E.K. were supported by the National Institute on Aging of the National Institutes of Health under Award Number R01AG038961. D.A. was supported by the NIH (R01EY033994; R61/33 HL159949; RF1 AG078352, R21NS130265) and by an unrestricted gift to the Division of Cerebrovascular Diseases and Stroke in the Department of Neurology, CUIMC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Author information
Authors and Affiliations
Contributions
T.K., D.A. and E.E.K. conceptualized the project, R.L.N., T.K., M.E.K., C.S.C, S.S., Y.N. and R.J. performed the experiments and analyzed the data, D.A. provided the transgenic mice, D.A. and E.E.K. provided guidance for the analysis of the data & T.K., R.L.N., D.A. and E.E.K. wrote and edited the manuscript.
Corresponding author
Ethics declarations
Competing interests
Some of the work presented is supported by patents optioned to Delsona Therapeutics, Inc. where EEK serves as a co-founder and scientific advisor. The remaining authors declare no competing interests.
Peer review
Peer review information
Communications Engineering thanks Wenlu Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: [Liangfei Tian] and [Philip Coatsworth]. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Noel, R.L., Kugelman, T., Karakatsani, M.E. et al. Safe focused ultrasound-mediated blood-brain barrier opening is driven primarily by transient reorganization of tight junctions. Commun Eng (2026). https://doi.org/10.1038/s44172-026-00597-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s44172-026-00597-5
