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Nitrous oxide enhances MR-guided focused ultrasound delivery of gene therapy to the murine hippocampus

Gene Therapy volume 32, pages 376–384 (2025)Cite this article

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Abstract

Transcranial Magnetic Resonance Guided Focused Ultrasound can oscillate intravenously delivered microbubbles and transiently open the blood brain barrier (BBB) in a targeted brain region. However, high microbubble doses or Focused ultrasound pressures (FUS) leads to injury. So, we administered nitrous oxide (N2O), an anesthetic gas to determine reduced need of FUS pressure and microbubble dose for opening BBB. Swiss Webster mice were treated with N2O or medical air (MA) at varying FUS pressures, while the microbubble dose was kept constant and the vice-versa. Consequently, BBB opening was quantified by acoustic emissions and enhancement rate on T1-weighted MR. To compare the effect of N2O on gene delivery, following BBB opening with either MA or N2O, a viral vector expressing GFP was subsequently delivered. Additionally, Immunohistochemical studies quantified viral transfection efficacy and assessed acute cell injury. We observed that N2O significantly potentiates acoustic emissions and enhancement rate on post-contrast MRI images, compared to MA at all measured pressures (0.39, 0.45, 0.67 MPa). Furthermore, N2O reduces the microbubble dose to 0.02μl/kg and FUS pressures to 0.28 and 0.39 MPa for BBB disruption and enhanced viral gene delivery, respectively. Hence, N2O potentiates microbubble oscillations, allowing reduced microbubble dose and FUS pressures and improved viral gene delivery.

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Fig. 1: Nitrous Oxide amplifies subharmonic acoustic Emissions, confirmed by T1W MRI image.
Fig. 2: Nitrous Oxide reduces FUS pressures required to open the Blood Brain Barrier.
Fig. 3: Nitrous Oxide reduces the microbubble dosage required to open BBB.
Fig. 4: Nitrous oxide enhances gene expression following FUS-mediated delivery of scAAV9/GFP to the hippocampus.
Fig. 5: Opening the BBB with Nitrous Oxide at lower pressures results in less injury than MA at a higher FUS pressures.

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Funding

UTSW High Impact award: BRS, RMB, RC.

Author information

Author notes

  1. These authors contributed equally: Deepshikha Bhardwaj, Ibrahim Youssef. Bhavya R. Shah is Senior Contributing Author and Corresponding author.

Authors and Affiliations

  1. Transcranial Focused Ultrasound Lab, UTSW Medical Center, Dallas, TX, USA

    Deepshikha Bhardwaj, Ibrahim Youssef, Darren Imphean, Venugopal Krishnan, Rajiv Chopra & Bhavya R. Shah

  2. Neuroradiology and Neuro-intervention Section, Department of Radiology, UTSW Medical Center, Dallas, TX, USA

    Deepshikha Bhardwaj, Ibrahim Youssef, Darren Imphean, Venugopal Krishnan, Rajiv Chopra & Bhavya R. Shah

  3. Department of Chemistry, Faculty of Science, Mansoura University, Dakahlia, Egypt

    Ibrahim Youssef

  4. Center for Alzheimer’s and Neurodegenerative Diseases, UTSW Medical Center, Dallas, TX, USA

    Sydni K. Holmes, Sandi Jo Estill-Terpack, Marc Diamond, Rachel M. Bailey & Bhavya R. Shah

  5. Department of Neurology, UTSW Medical Center, Dallas, TX, USA

    Marc Diamond

  6. Advanced Imaging Research Center, UTSW Medical Center, Dallas, TX, USA

    Rajiv Chopra & Bhavya R. Shah

  7. Department of Pediatrics, UTSW Medical Center, Dallas, TX, USA

    Rachel M. Bailey

  8. Department of Neurological Surgery, UTSW Medical Center, Dallas, TX, USA

    Bhavya R. Shah

  9. O’Donnell Brain Institute, UTSW Medical Center, Dallas, TX, USA

    Bhavya R. Shah

Authors
  1. Deepshikha Bhardwaj
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Contributions

BRS designed the work, performed experiments, prepared and revised manuscript; DB and IY performed experiments, manuscript preparation, and statistical analysis; DI manuscript preparation; VK, SKH, SJET, RC, RMB performed experiments and results interpretation; MD did manuscript preparation. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Corresponding author

Correspondence to Bhavya R. Shah.

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Competing interests

RC has competing interests with FUS Instruments. Other authors declare no competing interests.

Ethical approval

The animal protocol (101517) for this study was approved by the ethics committee of University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee (IACUC) and Confirmed to the National Institutes of Health’s PHS Policy on the Humane Care and Use of Laboratory Animals.

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Bhardwaj, D., Youssef, I., Imphean, D. et al. Nitrous oxide enhances MR-guided focused ultrasound delivery of gene therapy to the murine hippocampus. Gene Ther 32, 376–384 (2025). https://doi.org/10.1038/s41434-025-00530-z

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