Acute Inflammatory Response Following Increased Blood-Brain Barrier Permeability Induced by Focused Ultrasound is Dependent on Microbubble Dose.

Rationale: Focused ultrasound (FUS), in conjunction with circulating microbubbles (MBs), can be used to transiently increase the permeability of the blood-brain barrier (BBB) in a targeted manner, allowing therapeutic agents to enter the brain from systemic circulation. While promising preclinical work has paved the way for the initiation of 3 human trials, there remains concern regarding neuroinflammation following treatment. The aim of this study was to assess the magnitude of this response following sonication and explore the influence of MB dose. Methods: Differential expression of NFκB signaling pathway genes was assessed in rats at 6 h and 4 days following a FUS-mediated increase in BBB permeability. Three sonication schemes were tested: (1) a clinical imaging dose of MBs + peak negative pressure (PNP) controlled by acoustic feedback, (2) 10x clinical imaging dose of MBs + constant PNP of 0.290 MPa, and (3) 10x clinical imaging dose of MBs + PNP controlled by acoustic feedback. Follow-up magnetic resonance imaging (MRI) was performed to assess edema and hemorrhage. Hematoxylin and eosin histology was used to evaluate general tissue health. Results: MB dose has a significant impact on the expression of several key genes involved in acute inflammation and immune activation, including Tnf, Birc3, and Ccl2. At a clinical imaging dose of MBs, there were no significant changes detected in the expression of any NFκB signaling pathway genes. Conversely, a high MB dose resulted in a clear activation of the NFκB signaling pathway, accompanied by edema, neuronal degeneration, neutrophil infiltration, and microhemorrhage. Results also suggest that post-FUS gadolinium enhancement may hold predictive value in assessing the magnitude of inflammatory response. Conclusion: While a significant and damaging inflammatory response was observed at high MB doses, it was demonstrated that FUS can be used to induce increased BBB permeability without an associated upregulation of NFκB signaling pathway gene expression. This emphasizes the importance of employing optimized FUS parameters to mitigate the chances of causing injury to the brain at the targeted locations.

Introduction

Purpose Drug delivery with BBB opening

Study Objective To assess the magnitude of NFκB-mediated neuroinflammatory responses following focused ultrasound–induced blood–brain barrier opening and determine how microbubble dose influences that response.

Animal model / Human subject Rat (Rattus norvegicus), Sprague-Dawley strain, age not specified (weight 270–340 g at time of sonication), male

Disease model Healthy

MRI or image guidance method MRI-guided: targets chosen on T2-weighted MR images coregistered to the RK100 FUS positioning system (7-T MRI); contrast-enhanced T1-weighted images used for localization/LCM guidance; acoustic-emission feedback used for pressure control

Targeted brain region(s) Hippocampus

Cargo name and characteristics Gadobutrol (Gadovist) — a gadolinium-based small-molecule paramagnetic MRI contrast agent administered intravenously to assess blood–brain barrier permeability.

Route of administration Intravenous (tail vein catheter)

Outcomes and Safety

Summary of Outcomes High microbubble (MB) dose (10x clinical) during FUS induced NFκB-pathway activation accompanied by edema, neuronal degeneration, neutrophil infiltration and microhemorrhage, whereas a clinical imaging MB dose produced increased BBB permeability without upregulation of NFκB genes or histologic/MRI signs of injury. Of the sonication schemes tested, scheme 1 (clinical imaging MB dose + acoustic‑emissions‑controlled PNP) was successful/safe; schemes 2 (10x MB dose + constant PNP 0.290 MPa) and 3 (10x MB dose + acoustic‑feedback PNP) produced damaging inflammatory responses.

Duration of biological effect 4 days

Safety-related matter High microbubble (10x clinical) dose produced significant adverse effects including upregulation of NFκB inflammatory genes, MRI-detected edema and microhemorrhage, neuronal degeneration, RBC extravasation, leukocyte infiltration, gliosis and microglial nodules; by contrast, the clinical imaging MB dose increased BBB permeability without upregulation of NFκB genes or histological/MRI evidence of edema, hemorrhage, or tissue damage.

Brain Region

Ultrasound Parameters

Ultrasound instrument RK100 system (FUS Instruments Inc., Toronto, ON, Canada); spherically focused transducer — focal number 0.8; external diameter = 75 mm; internal diameter = 20 mm; frequency = 551.5 kHz

FUS Frequency 551.5 kHz

FUS Pressure 0.290 MPa; starting PNP 0.128 MPa (increased by 0.008 MPa every second); adaptive PNP reduced by 50% upon ultraharmonic threshold

FUS Mode pulsed

Pulse duration 10 ms

Duration of a single FUS session 2 minutes

Focal Characteristics focal depth: None; focal length: 60 mm; aperture size: 75 mm (external diameter; internal diameter 20 mm)

Treatment frequency Single session

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